Biological Amplification Research Articles

A hypothetical mathematical construct explaining the mechanism of biological amplification in an experimental model utilizing picoTesla (PT) electromagnetic fields

We seek to answer the conundrum: What is the fundamental mechanism by which very weak, low frequency Electromagnetic fields influence biosystems? In considering the hydrophobicity of intramembranous protein (IMP) H-bonds which cross the phospholipid bilayer of plasma membranes, and the necessity for photonic recycling in cell surface interactions after dissipation of energetic states, present models lack structure and thermodynamic properties to maintain (Δ E) sufficient energy sources necessary for amplifications by factors of 10 12. Even though one accepts that the ligand–receptor association alters the conformation of extracellular, extruding portions of IMP’s at the cell surface, and that this change can be transmitted to the cytoplasm by the transmembranous helical segments by nonlinear vibrations of proteins with generation of soliton waves, one is still unable to account for repair and balanced function. Indeed, responses of critical molecules to certain magnetic field signals may include enhanced vibrational amplitudes, increased quanta of thermal energies and order inducing interactions. We may accept that microtrabecular reticulum-receptor is associated with actin filaments and ATP molecules which contribute to the activation of the cyclase enzyme system through piezoelectricity. Magnetic fields will pass through the membrane which sharply attenuates the electric field component of an EM field, due to its high impedance. Furthermore, EM oscillations are converted to mechanical vibrations; i.e., photon–phonon transduction, to induce molecular vibrations of frequencies specifically responsible for bioamplifications of weak triggers at the membrane surface, as well as GAP junctions. The hydrogen bonds of considerable importance are those in proteins (10 12 Hz) and DNA (10 11 Hz) and may be viewed as centers of EM radiation emission in the range from the mm microwaves to the far IR. However, classical electrodynamical theory does not yield a model for biomolecular resonant responses which are integrated over time and account for the connection between the phonon field and photons. Jacobson Resonance does supply an initial physical mechanism, as equivalencies in energy to that of Zeeman Resonance (i.e., zero-order magnetic resonance) and cyclotron resonance may be derived from the DeBroglie wave particle equation. For the first time, we view the introduction of Relativity Theory to biology in the expression, mc 2=BvLq, where m is the mass of a particle in the ‘box’ or ‘string’ (molecule in a biosystem), c is the velocity of electromagnetic field in space, independent of its inertial frame of reference, B is the magnetic flux density, v is the velocity of the carrier or ‘string’ (a one or two dimensional ‘box’) in which the particle exists, L is its dimension (length) and q represents a unit charge q=1 C, by defining electromotive force as energy per unit charge. Equivalencies suggest that qvBL is one of the fundamental expressions of energy of a charged wave-particle in magnetic fields, just as Zeeman and cyclotron resonance energy expressions, gβB and qhB/2π m, and is applicable to all charged particles (molecules in biological systems). There may exist spontaneous, independent and incessant interactions of magnetic vector B and particles in biosystems which exert Lorentz forces. Lorentz forces may be transmitted from EM field to gravitational field as a gravity wave which return to the phonon field as microgravitational fluctuations to therein produce quantum vibrational states that increase quanta of thermal energies integrated over time. This may account for the differential of 10 12 between photonic energy of ELF waves and the Boltzman energy kT. Recent data from in vivo controlled studies are included as empirical support for the various hypotheses presented.

Homogeneous Assay for Tyrosine Kinase: Use of Bacteriophage Antibody Conjugates in an Assay for p56lck Kinase

Protein kinases play a critical role in almost every cellular regulatory process and have been identified as key players in diseases such as cancer and immune syndromes (1). For this reason, there has been substantial interest in the development of assays for protein kinases for use as both diagnostics and drug discovery tools (2). Although several assays exist for kinases, the most commonly used involve monitoring transfer of the γ-phosphoryl group from [γ-32P]ATP to a peptide substrate (3). These assays are cumbersome to implement because the unreacted [γ-32P]ATP needs to be separated from the phosphorylated peptide by use of separation techniques such as gel electrophoresis. In drug discovery programs, fluorescence resonance energy transfer (FRET) kinase assays are becoming increasingly popular because they are homogeneous, making them relatively easy to automate (4). In typical FRET assays, the anti-phosphotyrosine antibody and the substrate peptide are labeled with “donor” and “acceptor” fluorophores. On phosphorylation of the peptide, the anti-phosphotyrosine antibody binds to the peptide, and the two fluorophores are brought in close proximity to each other. Excitation of the acceptor fluorophore leads to energy transfer to the donor molecule, which emits fluorescence. The principal disadvantage of FRET-based assays is that they are difficult to configure because the two fluorophores need to be within a closely defined distance of each other. The Dual Phage technology is a new ultrasensitive biological amplification system of broad applicability that uses bacteriophages as …

Sobrevivência de Xanthomonas axonopodis pv. manihotis em manipueira sob condições ambientais

O objetivo deste trabalho foi avaliar a sobrevivência de um isolado de Xanthomonas axonopodis pv. manihotis (Xam), resistente ao sulfato de estreptomicina, em manipueira incubada em condições ambientais, em três épocas (março, julho e outubro de 2000). Foram utilizados um meio de cultura semi-seletivo para o isolamento de X. axonopodis pv. manihotis e a técnica de reação de polimerase em cadeia (PCR) ou de amplificação biológica (BIO-PCR). Foram determinados o pH e a acidez total da manipueira durante o período de incubação estudado. O isolado sobreviveu por um período inferior a 24 horas, nas três épocas do ano. A técnica de PCR ou BIO-PCR não foi eficaz em monitorar a sobrevivência do isolado em manipueira, pois apresentou resultados contraditórios e não confiáveis. Observou-se queda do pH e aumento da acidez total da manipueira incubada nas condições estudadas, que pode explicar o comportamento da sobrevivência de Xam em manipueira.

Experience with International Neuroblastoma Staging System and Pathology Classification

The International Neuroblastoma Staging System and Pathology Classification were proposed in 1988 and in 1999, respectively, but their clinical value has not yet been fully studied in new patients. Six hundred and forty-four patients with neuroblastoma treated between January 1995 and December 1999 were analysed by these classifications. The 4-year overall survival rate of patients <12 months of age with INSS stages 1, 2A, 2B, 3 and 4S disease was 98.5%, which was significantly higher than the 73.1% rate in stage 4 patients <12 months (P<0.0001). When patients were ⩾12 months, the 4-year overall survival rate of patients with neuroblastoma at 1, 2A, 2B and 3 stages was 100% and that of patients at stage 4 was 48.5% (P<0.0001). As to the International Neuroblastoma Pathology Classification histology, the 4-year overall survival rate was 98.8% in patients with favourable histology and 60.7% in those with unfavourable histology in the <12 months group (P<0.0001). In the ⩾12 months group, the 4-year oral survival of patients with favourable histology was 95.3% and that of patients with unfavourable histology was 50.6% (P<0.0001). Among biological factors, MYCN amplification, DNA diploidy and 1p deletions were significantly associated with poor prognosis in patients <12 months, as were MYCN amplification and DNA diploidy in patients ⩾12 months of age. Multivariate analysis showed that the INSS stage (stage 4 vs other stages) and International Neuroblastoma Pathology Classification histology (unfavourable vs favourable) were significantly and independently associated with the survival of patients undergoing treatment, stratified by age, stage and MYCN amplification (P=0.0002 and P=0.0051, respectively).

Experience with International Neuroblastoma Staging System and Pathology Classification.

The International Neuroblastoma Staging System and Pathology Classification were proposed in 1988 and in 1999, respectively, but their clinical value has not yet been fully studied in new patients. Six hundred and forty-four patients with neuroblastoma treated between January 1995 and December 1999 were analysed by these classifications. The 4-year overall survival rate of patients <12 months of age with INSS stages 1, 2A, 2B, 3 and 4S disease was 98.5%, which was significantly higher than the 73.1% rate in stage 4 patients <12 months (P<0.0001). When patients were ⩾12 months, the 4-year overall survival rate of patients with neuroblastoma at 1, 2A, 2B and 3 stages was 100% and that of patients at stage 4 was 48.5% (P<0.0001). As to the International Neuroblastoma Pathology Classification histology, the 4-year overall survival rate was 98.8% in patients with favourable histology and 60.7% in those with unfavourable histology in the <12 months group (P<0.0001). In the ⩾12 months group, the 4-year oral survival of patients with favourable histology was 95.3% and that of patients with unfavourable histology was 50.6% (P<0.0001). Among biological factors, MYCN amplification, DNA diploidy and 1p deletions were significantly associated with poor prognosis in patients <12 months, as were MYCN amplification and DNA diploidy in patients ⩾12 months of age. Multivariate analysis showed that the INSS stage (stage 4 vs other stages) and International Neuroblastoma Pathology Classification histology (unfavourable vs favourable) were significantly and independently associated with the survival of patients undergoing treatment, stratified by age, stage and MYCN amplification (P=0.0002 and P=0.0051, respectively).British Journal of Cancer (2002) 86, 1110–1116. DOI: 10.1038/sj/bjc/6600231 www.bjcancer.com© 2002 Cancer Research UK

Estandarización de la obtención de amplificados del genoma del virus de la rabia para su uso en estudios de epidemiología molecular

In order to improve the diagnosis and typification of rabies viruses at the Instituto Nacional de Salud National Reference Laboratory for rabies, we standardized techniques for the amplification of a 902 nucleotide DNA fragment, complementary to a selected region of the rabies virus genomic RNA. This region codes for a segment of both the glycoprotein and protein L, and contains the G-L intergenic noncoding region known as Pseudogen Psi. The standardized techniques included: 1) biological amplification of rabies viruses by intracerebral mouse inoculation; 2) total RNA extraction from the brains of infected mice, 3) RT-PCR amplification of a 902 nucleotide DNA fragment complementary to the selected RNA region. The study sample consisted of 30 rabies virus strains isolated from dogs and selected from the virus bank of the Virology Laboratory. Due to their simplicity the methods described have several advantages when compared to methods reported in previous papers. The technology proposed is a precise complement to rabies diagnosis techniques, and can be applied to the identification of phylogenetic relations among rabies isolates, and, therefore, it is also used to identify rabies transmission dynamics and geographical distribution.

Combined bisulfite restriction analysis (COBRA).

AbstractMost molecular biological techniques used to analyze specific loci in complex genomic DNA involve some form of sequence-specific amplification, whether it is biological amplification by cloning in Escherichia coli, direct amplification by polymerase chain reaction (PCR), or signal amplification by hybridization with a probe that can be visualized. Since DNA methylation is added postreplicatively by a dedicated maintenance DNA methyltransferase that is not present in either E. coli or in the PCR reaction, the methylation information is lost during molecular cloning or PCR amplification. Molecular hybridization does not discriminate between methylated and unmethylated DNA, since the methyl group on the cytosine does not participate in base pairing. The lack of a facile way to amplify the methylation information in complex genomic DNA has been a significant impediment to DNA methylation research.KeywordsPolymerase Chain ReactionPolymerase Chain Reaction ProductPolymerase Chain Reaction AmplificationPolymerase Chain Reaction ReactionSodium BisulfiteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Lentiviral vectors approach the clinic but fall back: National Institutes of Health Recombinant DNA Advisory Committee review of a first clinical protocol for use of a lentiviral vector.

Lentiviral vectors approach the clinic but fall back: National Institutes of Health Recombinant DNA Advisory Committee review of a first clinical protocol for use of a lentiviral vector.

Single-molecule immunoassay and DNA diagnosis.

Many assays relevant to disease diagnosis are based on electrophoresis, where the migration velocity is used for distinguishing molecules of different size or charge. However, standard gel electrophoresis is not only slow but also insensitive. We describe a single-molecule imaging procedure to measure the electrophoretic mobilities of up to 100000 distinct molecules every second. The results correlate well with capillary electrophoresis (CE) experiments and afford confident discrimination between normal (16.5 kbp) and abnormal (6.1 kbp) mitochondrial DNA fragments, or beta-phycoerythrin-labeled digoxigenin (BP-D) and its immunocomplex (anti-D-BP-D). This demonstrates that virtually all electrophoresis diagnostic protocols from slab gels to CE should be adaptable to single-molecule detection. This opens up the prossibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction (PCR) or other biological amplification.

High-throughput single molecule screening of DNA and proteins.

We report a novel imaging technology for real time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g., DNA region, RNA region, protein), and to do so at a relatively low cost. The technology is suitable for DNA and RNA through sizing or through fluorescent hybridization probes, and for proteins and small molecules through fluorescence immunoassays. This combination of the lowest possible detection limit and the broadest applicability to biomolecules represents the final frontier in bioanalysis. The general scheme is based on novel concepts for single molecule detection (SMD) and characterization recently demonstrated in our laboratory. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. This opens up the possibility of finding single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification.

High-throughput single-molecule DNA screening based on electrophoresis.

In electrophoresis, the migration velocity is used for sizing DNA and proteins or for distinguishing molecules based on charge and hydrodynamic radius. Many protein and DNA assays relevant to disease diagnosis are based on such separations. However, standard protocols are not only slow (minutes to hours) but also insensitive (many molecules in a detectable band). We successfully demonstrated a high-throughput imaging approach that allows determination of the individual electrophoretic mobilities of many molecules at a time. Each measurement only requires a few milliseconds to complete. This opens up the possibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. The purpose is not to separate the DNA molecules but to identify each one on the basis of the measured electrophoretic mobility. We developed three different procedures to measure the individual molecular mobilities. The results correlate well with capillary electrophoresis (CE) experiments for the same samples (2-49 kb dsDNA) under identical separation conditions. The implication is that any electrophoresis protocols from slab gels to CE should be adaptable to single-molecule screening for disease diagnosis.

Redfield revisited: 2. What regulates the oxygen content of the atmosphere?

The continuous charcoal record, interpreted with the aid of the results of combustion experiments, indicates that the mixing ratio of atmospheric oxygen has varied remarkably little over the past 350 Myr. We develop a dynamic feedback model of the coupled P, N, C, and O2 cycles and use perturbation analysis and a case study of the past 40 Myr to test various feedback mechanisms that have been proposed to stabilize atmospheric oxygen. These mechanisms involve alterations in nutrient driven productivity and the subsequent burial flux of organic carbon, which provides the main source of atmospheric oxygen. Suppression of the burial of phosphorus sorbed to iron minerals under anoxic conditions in ocean bottom waters tends to increase the ocean nutrient inventory and provide negative feedback against declining oxygen [Holland, 1994]. However, denitrification is enhanced by anoxia, tending to reduce the nutrient inventory and amplify declining oxygen [Lenton and Watson, this issue]. If organic phosphorus removal from the ocean is also suppressed under anoxic conditions, this improves oxygen regulation [Van Cappellen and Ingall, 1994], as does direct enhancement of organic carbon burial due to reduced oxygen concentration in bottom waters [Betts and Holland, 1991]. However, all of the ocean‐based feedback mechanisms cease to operate under increases in oxygen that remove anoxia from the ocean. Fire frequency is extremely sensitive to increases in oxygen above 21% of the atmosphere, readily suppressing vegetation on the land surface. This should transfer phosphorus from the land to the ocean, causing less carbon to be buried per unit of phosphorus and providing a weak negative feedback on oxygen [Kump, 1988]. However, a new proposal that increases in oxygen suppress the biological amplification of rock weathering and hence the input of phosphorus to the Earth system provides the most effective oxygen regulation of all the mechanisms considered. A range of proxies suggests that the input of available phosphorus to the ocean may have been significantly reduced 40 Myr ago, suppressing new production and organic carbon burial in the model. With only ocean‐based feedback, the atmospheric oxygen reservoir is predicted to have shrunk from ∼26% of the atmosphere 40 Myr ago. However, when land plant mediated negative feedback on phosphorus weathering is added, oxygen is regulated within 19–21% of the atmosphere throughout the past 40 Myr, in a manner more consistent with paleorecords.

Anatomy of an Endogenous Antagonist: Relationship between Agouti-Related Protein and Proopiomelanocortin in Brain

Agouti-related protein (AGRP) is a recently discovered orexigenic neuropeptide that inhibits the binding and action of alpha-melanocyte-stimulating hormone derived from proopiomelanocortin (POMC) at the melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) and has been proposed to function primarily as an endogenous melanocortin antagonist. To better understand the interplay between the AGRP and melanocortin signaling systems, we compared their nerve fiber distributions with each other by immunohistochemistry and their perikarya distribution with MC3R and MC4R by double in situ hybridization. Although deriving from distinct cell groups, AGRP and melanocortin terminals project to identical brain areas. Both AGRP and melanocortin neurons selectively express the MC3R, which provides a neuroanatomical basis for a dual-input circuit with biological amplification and feedback inhibition. These studies highlight a broader complexity in POMC-mediated behavior in the brain.

Comparison of PCR, BIO‐PCR, DIA, ELISA and isolation on semiselective medium for detection of Xanthomonas albilineans, the causal agent of leaf scald of sugarcane

Polymerase chain reaction (PCR) and newly designed primers, XAF1/XAR1, were tested for selective detection of the causal agent of leaf scald of sugarcane, Xanthomonas albilineans. The efficiency and reliability of PCR were compared with dot immunobinding assay (DIA), ELISA and classical isolation techniques for detecting X. albilineans in suspensions of pure cells and extracts of field‐collected stalk and leaf samples of sugarcane. In addition, classical PCR and BIO‐PCR (biological amplification followed by PCR) were compared with isolation on a semiselective agar medium. Classical PCR and BIO‐PCR techniques had the advantage of not requiring pathogenicity tests to confirm the identity of colonies tentatively identified as X. albilineans on modified semiselective XAM agar medium. The m‐XAM medium and BIO‐PCR techniques were the most sensitive; however, the former required seven days whereas the latter required only four days. The BIO‐PCR technique was as sensitive as the semiselective medium technique and eliminated the need to conduct any additional tests to confirm the identification.

In vitro selection of aptamers from RNA libraries.

Methods of iterative nucleic acid selection and amplification were enabled by the invention of the polymerase chain reaction (PCR). Thus, the ability to amplify as few as a single DNA or RNA molecule made it possible to diversify sequences and to partition the desirable from the undesirable subset (Fig. 1A) (,). Initially, this approach was practiced in vivo using biological amplification and selection, by diversification of plasmid sequences and iterative growth against a selective marker (). The power of in vitro RNA selection from a randomized combinatorial library was demonstrated by Tuerk and Gold () using T4 DNA polymerase and the R17 phage coat protein. These investigators called their iterative RNA selection procedure “SELEX.” Ellington and Szostak () also derived RNA ligands against organic dyes using iterative in vitro selection. Moreover, the demonstration that RNAs could be selected that bind to proteins and compounds with no known role in RNA-binding in vivo led to the concept of aptamers. An aptamer is a folded RNA that forms a shape that coincidentally fits against another surface, to which it is “apt” to bind. Generally, RNA ligands that bind to naturally occurring RNA-binding domains of proteins are not considered to be aptamers. Unexpectedly, an RNA ligand selected against an antibody generated by immunization with a peptide demonstrated that RNA and protein could crossreact at the level of antibody recognition (). Open image in new window Fig. 1A. (A) A schematic representation of the randomized RNA selection protocol. The sequences of the primers and template are given in (B). The T7 promoter region is labeled T7 pro. DNA and RNA are represented by double lines and single lines, respectively. The number of iterative cycles used varies with the nature of the target molecule and can allow evolution of the target set using error-prone PCR.

Surviving the limits to life at the surface of Mars

The harshness of the environment at its surface severely limits the survival of unprotected terrestrial organisms on Mars. However, the Martian geologic record is consistent with an earlier history with much different climatic conditions, more conducive to life, followed by an extended climate change which led to the currently inhospitable state. Such a transition is ideal for evolutionary adaptation to new conditions because it provides both the forcing function and the extensive time for progressive specializations. It is shown that against each of the current limits to life (organics deprivation; restricted amounts and physical forms of H2O; limited availability of other key nutrients; ionizing radiation insult; and strongly oxidizing chemical species), there are plausible countermeasure solutions. Most, but not all, of these mechanisms already exist as known examples in terrestrial biota. A combination of these adaptations could conceivably allow highly tailored organisms, either active or dormant, to survive at or very near the surface of Mars. Even in carefully preserved regolith samples returned to Earth, microorganisms could be difficult to detect without specific biochemical probes and/or purposeful biological amplification by selective enhancement of the supply of nutrients, optimized incubation parameters, and highly sensitive detection of metabolic activity.

A high-throughput hybridization method for titer determination of viruses and gene therapy vectors.

One of the challenges facing researchers working with viruses and gene therapy vectors is the need to rapidly assay for infectious virus. Current methods used to titer many viruses are cumbersome and are not amenable to handling large numbers of samples. Here we describe the development of an assay that can rapidly quantify infectious viruses and gene therapy vectors. The assay relies on biological amplification of viral sequences and hybridization of labeled probes to immobilized nucleic acid from infected cells. The amplification of the viral genome makes this a highly sensitive method. The assay is configured in a high-throughput format that has been used to detect recombinant adeno-associated virus (AAV), wild-type AAV and infectious adenovirus. The assay is quantitative, and can be used to titer virus preparations with or without a known standard.

PCR‐Based Detection of Artificial Latent Infections of Geranium by Xanthomonas campestris pv. pelargonii

AbstractA procedure entailing biological enrichment and PCR amplification was developed to detect small populations of Xanthomonas campestris pv. pelargonii (X.c, pv. pelargonii) in tissues of geranium. Known numbers of colony forming units (CFU) of X.c. pv. pelargonii were introduced into ‘Red Elite’ geraniums through wounding of petioles and stems. Immediately after inoculation, sections of the petioles and stems were harvested and incubated for 24 or 48 h in nutrient broth (biological enrichment). After enrichment, bacterial cells were collected by centrifugation, followed by rapid extraction of total nucleic acid from the cells with GeneReleaser™, PCR amplification of DNA with pathovar‐specific primers, and ethidium bromide‐stained agarose gel electrophoretic analysis of the PCR products. After 48 h biological enrichment, it was possible to detect as few as 1 CFU of Xc. pv. pelargonii in stems and petioles collected immediately after inoculation, with the detection limit ranging between 1 and 120 CFU during multiple experiments. It also was possible to detect systemic movement of the bacterium in intact plants sampled 24 h after inoculation with a minimal inoculum (4 CFU). This procedure may have application in geranium certification programs concerned with the detection of latent infections associated with low levels of X.c.pv. pelargonii.

Rapid and specific diagnosis of group B streptococcal infection by the polymerase chain reaction (PCR).

Polymerase Chain Reaction (PCR) has proved its importance in the field of diagnostic molecular biology. By replacing biological amplification (growth on culture media) with enzymatic amplification of nucleotide sequences specific to microbes PCR significantly shortens elapsed time involved to detect and identify a microbe responsible for causing an infection.

Mechanical responses of the basilar membrane.

The traveling‐wave activity that Georg von Bekesy studied in the cochlea early in this century has been of intense interest to physiologists and mathematical modelers over the last 30 years. This interest was nurtured by the discovery that the mechanical responses of the basilar membrane are nonlinear at relatively low sound levels and that the nonlinearity is dependent on biological processes [W. S. Rhode, J. Acoust. Soc. Am. Suppl. 1 49, S1218 (1971)]. This review will briefly summarize the major findings of the early years as background to a presentation of several issues that are the focus of contemporary work. The steady‐state and transient velocity responses of the basilar membrane characterize the capacity of the biological amplification mechanism in the organ of Corti and indicate that a ‘‘gain’’ of 40–60 dB is provided by the outer hair cells. Considerable response distortion accompanies this gain, and wave propagation of certain distortion products occurs. Electrical stimulation of the outer hair cells provides evidence of their mechanical role in the amplification mechanism by demonstration of the frequency range and displacement capacity of the cells. Efferent innervation of the organ of Corti is found to modulate the mechanical activity of the system.