Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part II: Oncology, chemotherapy and carcinogenesis.
This paper follows on from Part 1 of my review of bromodeoxyuridine published earlier this year (Dolbeare, 1995).
Animals↗
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Results for “tool biologic”
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Recent advances on proteins of plant terminal membranes.
Since the beginning of the 1990s, our knowledge of the protein equipment of plant membranes progresses at an accelerating pace, owing to the irruption of molecular biology tools and genetics strategies in plant biology. Map-based cloning strategies and exploration of EST databases rapidly enrich the catalog of cDNA or gene sequences expected to code for membrane proteins. The accumulation of 'putative' membrane proteins reinforces the need for structural, functional and physiological information. Indeed, ambiguities often exist concerning the association to a membrane, the membrane identity and the topology of the protein inserted in the membrane. The combination of directed mutagenesis and heterologous expression of plant genes in various systems and plant reverse genetics has opened the possibility to study molecular and physiological functions. This review will emphasize how these tools have been essential for the exciting recent discoveries on plant terminal membrane proteins. These discoveries concern a variety of transport systems for ions, organic solutes including auxin, water channels, a large collection of systems suspected to act as receptors of chemical signals, proteins thought to control vesicle trafficking and enzymatic systems.
Animals↗
Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I/II pocket.
RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis in vivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose in vivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.
Biological Products↗
The ERATO Systems Biology Workbench: enabling interaction and exchange between software tools for computational biology.
Researchers in computational biology today make use of a large number of different software packages for modeling, analysis, and data manipulation and visualization. In this paper, we describe the ERATO Systems Biology Workbench (SBW), a software framework that allows these heterogeneous application components--written in diverse programming languages and running on different platforms--to communicate and use each others' data and algorithmic capabilities. Our goal is to create a simple, open-source software infrastructure which is effective, easy to implement and easy to understand. SBW uses a broker-based architecture and enables applications (potentially running on separate, distributed computers) to communicate via a simple network protocol. The interfaces to the system are encapsulated in client-side libraries that we provide for different programming languages. We describe the SBW architecture and the current set of modules, as well as alternative implementation technologies.
Computational Biology↗
Can autologous bone culture predict spinal fusion capacity?
The capacity of the individual patient to initiate osteoblast proliferation as a predictor for successful lumbar spinal fusion has not yet been reported. The objectives of this study were, first, to analyze the relationship between in vitro osteoblast proliferation and clinical bony fusion in the individual patient in order to predict the fusion outcome and, second, to measure the effect of preoperative tobacco smoking on osteoblast proliferation. Sixty-one patients (mean age 46 years) underwent posterolateral lumbar fusion in the period 1994-1995. Thirty-eight patients received CD pedicle screw implants and 23 received posterolateral fusions alone. During surgery, autogenous iliac bone was harvested and 1 g of trabecular bone without blood or bone marrow was then isolated for cell culturing. The cultures were classified as excellent (confluence within 4 weeks), good (confluence between 4 and 6 weeks) and poor (no or poor growth). Spine fusion was evaluated by two independent observers from plain anterior-posterior, lateral, and flexion/extension radiographs taken 1 year postoperatively, and the functional outcome was measured by the Dallas Pain Questionnaire (DPQ). Twenty-three patients had excellent, 19 good, and 19 poor in vitro osteoblast proliferation. Bony fusion was obtained in 77% of patients: 83% in the CD instrumentation group and 70% in the non-instrumentation group (NS). There was no significant correlation between osteoblast proliferation and spinal fusion or functional outcomes when analyzing the CD instrumentation and non-instrumentation groups together or separately. Elderly patients had a significantly poorer osteoblast proliferation than younger patients (P < 0.008). Preoperative tobacco consumption had no discernible effect on osteoblast proliferation, and no correlation between smoking and fusion was found. Further refinement of autologous osteoblast culturing may provide a biological tool for selection of patients who require biological enhancement of their bone fusion capacity. The poorer osteoblast proliferation related to advanced age supports the important negative biological influence of age on bony fusion. However, with more sensitive testing and better discrimination, other results are possible - or can in any event not be excluded.
Adult↗
Molecular and cellular adaptation of muscle in response to physical training.
Molecular biology tools can be used to answer questions as to how adaptations occur in skeletal muscle with training that could provide new frameworks to improve physical performance. A number of mRNAs for transfer of metabolic substrates into muscle cells increase after a single bout of exercise demonstrating the responsiveness of some gene expression to exercise. In stretch-induced hypertrophy SRE1 of the skeletal alpha-actin promoter is required to transactivate the promoter. Less retardation of SRF in crude nuclear extracts from the stretched muscle implies a conformational change in SRF because of the stretch. Transgenic animals will provide a tool to test questions concerned with how exercise signals adaptive changes in gene expression. Molecular biological approaches will be able to evaluate the interaction between physical activity levels and the expression of genes that modulate the susceptibility to many chronic diseases. Benefits of exercise extend beyond fitness to better health. Molecular biology is an important tool which should lead to improved physical performance and health in both elite athletes and the general public.
Adaptation, Physiological↗
The purines: potent and versatile small molecule inhibitors and modulators of key biological targets.
The goal of this review is to highlight the wide range of biological activities displayed by purines, with particular emphasis on new purine-based agents which find potential application as chemical-biology tools and/or therapeutic agents. The expanding interest in the biological properties of polyfunctionalized purine derivatives issues, in large part, from the development of rapid high-throughput screening essays for new protein targets, and the corresponding development of efficient synthetic methodology adapted to the construction of highly diverse purine libraries. Purine-based compounds have found new applications as inducers of interferon and lineage-committed cell dedifferentiation, agonists and antagonists of adenosine receptors, ligands of corticotropin-releasing hormone receptors, and as inhibitors of HSP90, Src kinase, p38alpha MAP kinase, sulfotransferases, phosphodiesterases, and Cdks. The scope of application of purines in biology is most certainly far from being exhausted. Testing purine derivatives against the multitude of biological targets for which small molecule probes have not yet been found should thus be a natural reflex.
Antiviral Agents↗
General principles of DNA repair in microorganisms and implications for future research.
The analysis of DNA repair and mutagenesis in organisms that have high resolution genetic and molecular biological systems has led, and will continue to lead, to major advances in our understanding of these processes. It is striking that almost all of the major insights highlighted in this chapter have been gained within the last 10 years and, in fact, several of the discoveries described here occurred during the actual writing of this report. It is worth reflecting that in the mid 1970s there was a widely held view that the essential elements of DNA repair in E. coli were well understood and, at that time, a considerable number of investigators shifted their focus to systems that did not offer the high resolution genetic and molecular biologic tools of E. coli and S. cerevisiae. The remarkable progress achieved over the past ten years underscores the limitations of that viewpoint. It is difficult, and probably unwise, to pinpoint specific issues in DNA repair in E. coli and S. cerevisiae as special targets for future research since so much remains to be learned. Most of the striking discoveries outlined in this chapter have come from skilled investigators using the full range of high resolution genetic, molecular biologic, and biochemical tools to address fundamental problems in DNA repair and mutagenesis. In most cases, it would have been impossible to have predicted the critical discoveries that have been made. For example, UV and chemical mutagenesis require the induction of a pair of genes, mediated by DNA-damage-stimulated proteolytic cleavage of a repressor and then the subsequent activation of one of these gene products by a mechanistically related proteolytic cleavage. In 1975 an hypothesis proposing such a mechanism for UV and chemical mutagenesis would have been dismissed as being utterly fanciful. Yet this is the current reality. As in the cases of the other findings detailed here, the key insights came not from the testing of fanciful hypotheses but rather from systematic scientific studies of fundamental problems. Funding in this area should then support inquiries of fundamental issues in DNA repair and mutagenesis by investigators who are skilled at using the full range of sophisticated experimental tools available for these organisms. It is worth noting that supporting this type of research will shed light not only on mechanisms of DNA repair and mutagenesis but also on more fundamental issues in the biology of living organisms.(ABSTRACT TRUNCATED AT 400 WORDS)
Adaptation, Physiological↗
[Tools of molecular biology, what can be expected from them?].
The techniques of molecular biology represent a new tool for research and diagnosis. They have been used routinely since the middle 80s. The first applications were mainly in hereditary diseases. These techniques investigated an information and not direct biological activity and so new strategies had to be developed. Two techniques have played key roles. They are Southern's method which enables the detection of specific sequences among DNA fragments in any individual within a few days, and the method of in vitro selective amplification (PCR) which is the equivalent of cloning a sequence of several hundred pairs of bases in any individual in less than 3 hours. In hereditary diseases, molecular biological techniques enable diagnosis of the genetic abnormality responsible for the condition, even when the defect is not known. To this end, two strategies are available. The first, and the most satisfactory, is the direct strategy of characterising the genetic defect itself. This is possible when the mutation is a major alteration of the DNA molecule such as a deletion, an insertion or a recombination or when the mutation is isolated and known. In other cases, an indirect approach may be used which consists of determining whether the subject has received normal or defective chromosomes from his or her parents. The identification of good or bad chromosomes is based on the study of DNA markers: polymorphism. The indirect strategy can only be used under certain conditions: presence of an index case, informativity of polymorphisms, complete family study.(ABSTRACT TRUNCATED AT 250 WORDS)
Blotting, Southern↗
Comparison of the diagnostic and prognostic value of biological markers in neuroblastoma. Proposal for a common methodology of analysis. SENSE group.
BACKGROUND: The prognosis of pediatric neuroblastoma depends both on clinical presentation and on certain cellular and molecular characteristics. Screening programs have been initiated in infants of less than one year of age, based on the hypothesis that neuroblastoma progresses from early to late clinical stages through a classical multistep process linked to an accumulation of molecular abnormalities. However, recent analyses suggest that most cases discovered by screening are low stage tumors considered as dysembryogenetic residues devoid from major abnormalities and that high-grade tumors with molecular abnormalities are unrelated diseases. AIM OF THE REVIEW: To confirm one or the other hypothesis, and eventually identify biological factors possibly responsible for the initiation and progression of the disease, it is of utmost importance that all investigators agree on biological criteria for analysis when neuroblastoma tissue is available in screened and unscreened populations. This paper reviews the biological tools available for prognosis in neuroblastoma, the priority for analysis of biological markers according to both methodological reliability and feasibility, and the conditions of tissue storage for further analysis of these biological markers. CONCLUSION: The standardized biological evaluation of neuroblastoma will allow to collect sufficient data for multivariate analysis; such analysis is now fundamental if one wants to clearly define the respective impacts of biological abnormalities on neuroblastoma progression.
Biomarkers, Tumor↗
[Biological markers for the prognosis of neuroblastoma: proposal of a method of analysis].
The prognosis of pediatric neuroblastoma depends both on clinical presentation and on certain cellular and molecular characteristics. At the present time, two hypotheses can be drawn to explain both clinical and biological heterogeneity. In the first hypothesis, neuroblastoma progresses from early to late clinical stages through a classical multistep process linked to an accumulation of molecular abnormalities. In the second hypothesis, neuroblastoma represents an heterogeneous group of unrelated diseases, where most of stages I and II or stage IVS neuroblastomas can rather be considered as benign tumors, and stage IV neuroblastoma as a true malignant proliferation. To ascertain relevant biological factors for the prognosis of the disease, it is uppermost important that all investigators agree on biological criteria for analysis when neuroblastoma tissue is available in screened and unscreened populations. This paper reviews the biological tools available for prognosis in neuroblastoma, the priority for analysis of biological markers according to reliability, feasibility, and reproducibility of analysis procedure, and the conditions of tissue storage for further analysis of these biological markers. The standardized biological evaluation of neuroblastoma will allow, first, to collect sufficient data for multivariate analysis of prognostic factors and, second, to better define the putative links between various forms of the disease.
Biomarkers, Tumor↗
Practical bioinformatics for proteomics.
We used various bioinformatic tools to examine the unknown protein gi|12841975 that was up-regulated in mouse diabetic kidneys. The data indicate that this unknown protein is, indeed, the PEBP. Motif scanning showed that this protein contains several kinase motifs, especially PKC that plays an important role in the pathogenesis of diabetic nephropathy [24, 25]. We therefore hypothesize that this protein (PEBP) has a potential functional role in PKC-dependent pathogenic pathways of diabetic nephropathy. Further study will be focused on phosphorylation pathways of the PEBP and its substrates. In summary, we have presented a case study that outlines our approach to further characterize the unknown proteins identified by peptide mass fingerprinting. Publicly accessible bioinformatic tools can provide a wealth of information to guide subsequent approaches that use traditional molecular biology tools.
Animals↗
Genotype characterization of the bacterium expressing the male-killing trait in the ladybird beetle Adalia bipunctata with specific rickettsial molecular tools.
The male-killing ladybird beetle (LB) bacterium (AB bacterium) was analyzed with specific rickettsial molecular biology tools in the LB Adalia bipunctata strains. Eight phenotype-positive LB strains showing mortality of male embryos were amplified with rickettsial genus-specific primers from the gene for citrate synthase (CS) and the gene for a 17-kDa protein and spotted fever group-specific primers from the gene for the 120-kDa outer membrane protein (ompB). The specificity of amplification was confirmed by Southern hybridization and the absence of the above-listed gene products in three phenotype-negative LB strains. Restriction polymorphism patterns of three examined amplicons from the CS gene, 17-kDa-protein gene, and ompB gene were identical among the eight phenotype-positive LB strains and were unique among all known rickettsiae of the spotted fever and typhus groups. Amplified fragments of the CS genes of the AB bacterium, Rickettsia prowazekii Breinl, Rickettsia typhi Wilmington, Rickettsia canada 2678, and Rickettsia conorii 7 (Malish) were sequenced. The greatest differences among the above-listed rickettsial and AB bacterium CS gene sequences were between bp 1078 and 1110. Numerical analysis based on CS gene fragment sequences shows the close relationships of the AB bacterium to the genus Rickettsia. Expanding of knowledge about rickettsial arthropod vectors and participation of rickettsiae in the cytoplasmic maternal inheritance of arthropods is discussed.
Amino Acid Sequence↗
2004 ASM Conference on the New Phage Biology: the 'Phage Summit'.
In August, more than 350 conferees from 24 countries attended the ASM Conference on the New Phage Biology, in Key Biscayne, Florida. This meeting, also called the Phage Summit, was the first major international gathering in decades devoted exclusively to phage biology. What emerged from the 5 days of the Summit was a clear perspective on the explosive resurgence of interest in all aspects of bacteriophage biology. The classic phage systems like lambda and T4, reinvigorated by structural biology, bioinformatics and new molecular and cell biology tools, remain model systems of unequalled power and facility for studying fundamental biological issues. In addition, the New Phage Biology is also populated by basic and applied scientists focused on ecology, evolution, nanotechnology, bacterial pathogenesis and phage-based immunologics, therapeutics and diagnostics, resulting in a heightened interest in bacteriophages per se, rather than as a model system. Besides constituting another landmark in the long history of a field begun by d'Herelle and Twort during the early 20th century, the Summit provided a unique venue for establishment of new interactive networks for collaborative efforts between scientists of many different backgrounds, interests and expertise.
Bacteria↗
The impact of molecular biology on neuroscience.
How our brains work is one of the major unsolved problems of biology. This paper describes some of the techniques of molecular biology that are already being used to study the brains of animals. Mainly as a result of the human genome project many new techniques will soon become available which could decisively influence the progress of neuroscience. I suggest that neuroscientists should tell molecular biologists what their difficulties are, in the hope that this will stimulate the production of useful new biological tools.
Animals↗
Molecular parasitology goes cellular.
Application of molecular genetics is an increasingly feasible undertaking in parasitology; however, an understanding of the phenotypes of various mutants generated by this approach will ultimately rely on cellular approaches to resolve the function of various proteins. In addition to the advances in understanding the biology of parasitism, such studies probably will uncover fundamental aspects of how eukaryotic cells communicate and regulate their responses to the environment. Thus, parasites may one day provide useful biological tools, just as viruses and bacterial toxins have done in the past, towards probing the biology of mammalian cells.
Animals↗
16S ribosomal RNA tools identify an unexpected predominance of Paenibacillus-like bacteria in an industrial activated sludge system suffering from poor biosolids separation.
Molecular biology tools targeting 16S ribosomal RNA (16S rRNA) were used to identify a predominant bacterial population in a full-scale dairy wastewater activated sludge system suffering from poor biosolids separation. Gram and acridine orange staining indicated that viable, Gram-positive microorganisms were present in samples removed from the influent waste stream and represented approximately 50% of total cell counts in samples removed from the mixed liquor. Subsequently, the "full-cycle 16S rRNA approach" showed that phylogenetic relatives of Paenibacillus spp., a low guanine-plus-cytosine percent DNA-content, Gram-positive microorganism, represented up to 30% of total 4,6-diamidino-2-phenylindole (DAPI)-stained cell counts in samples of mixed liquor. Although fluorescent in situ hybridizations with 16S rRNA-targeted oligonucleotide hybridization probes identified Paenibacillus-like spp. in samples removed from the influent waste stream, their abundance was less than 10% of total stained cell counts. Results of this study suggest that Paenibacillus-like spp. were present in low abundance in the influent waste stream, increased in relative abundance within the treatment system, and should be examined further as a candidate bacterial population responsible for poor biosolids separation. This study demonstrates that the full-cycle 16S rRNA approach can be used to identify candidate bacterial populations that may be responsible for operational upsets in full-scale activated sludge systems without prior information from cultivation or microscopic analyses.
Bacillaceae↗
Na+/H+ antiporters, molecular devices that couple the Na+ and H+ circulation in cells.
Na+/H+ antiporters are universal devices involved in the Na+ and H+ circulation of both eukaryotes and prokaryotes, thus playing an essential role in the pH and Na+ homeostasis of cells. This review focuses on the major impact of the application of molecular biology tools in the study of the antiporters. These tools permit the verification of the role of the antiporters and provide insights into their unique biology. A novel signal transduction to Na+ involving nhaR, a positive regulator, controls the expression of nhaA in E. coli. A "pH sensor" regulates the activity of Na+/H+ antiporters, both in eukaryotes and prokaryotes. A most intricate signal transduction to pH involving phosphorylation steps controls the activity of nhel in higher mammals. The identification of Histidine 226 in the "pH sensor" of NhaA is a step forward towards the understanding of the pH regulation of these proteins.
Animals↗