Mechanical force-based probing of intracellular proteins from living cells using antibody-immobilized nanoneedles

We developed a method combining atomic force microscopy (AFM) and antibody-immobilized nanoneedles to discriminate living cells by probing intracellular cytoskeletal proteins without the need for cell labeling. The nanoneedles are ultra-thin AFM probes sharpened to 200 nm in diameter. While retracting a nanoneedle inserted into a cell, we measured the mechanical force needed to unbind the antibody-target protein complex. Using this method, the intermediate filament protein, nestin and neurofilament were successfully detected in mouse embryonic carcinoma P19 cells and rat primary hippocampal cells within a minute for a single cell and cell differentiation states could be determined. Additionally, the measured magnitude of the force detecting nestin was indicative of the malignancy of breast cancer cells. This method was shown to affect neither the doubling time of cells nor does it leave extrinsic antibodies within the examined cells, allowing to be used in subsequent analyses in their native state.     

Quantifying the relationship between multiple immunological parameters and host resistance: probing the limits of reductionism

Although reductionist experimental designs are excellent for identifying cells, molecules, or functions involved in resistance to particular microbes or cancer cells, they do not provide an integrated, quantitative view of immune function. In the present study, mice were treated with either dexamethasone (DEX) or cyclosporin A (CyA), and immune function and host resistance were evaluated. Multivariate statistical methods were used to describe the relative importance of a broad range of immunological parameters for host resistance in mice treated with various dosages of DEX. Multiple regression and logistic regression analysis indicated that changes in 24 immunological parameters explained a substantial portion of the changes in resistance to B16F10 tumor cells or streptococcus group B. However, at least 40% of the change in host resistance remained unexplained. DEX at all dosages substantially suppressed numerous relevant immunological parameters, but significantly decreased resistance to Listeria monocytogenes only at the highest dosage. In contrast, CyA substantially decreased resistance to L. monocytogenes at dosages that caused relatively minor suppression of just a few immunological parameters (unfortunately, CyA data and host resistance data for L. monocytogenes were not suitable for multivariate analysis). These results illustrate that mathematical models can be used to explain changes in host resistance on the basis of changes in immune parameters, and that moderate changes in relevant immunological parameters may not produce the types of changes in host resistance expected on the basis of results from reductionist experimental designs.     

Advances in the study of luminescence probes for proteins

Spectral probes (or labels) have been widely used for the investigation and determination of proteins and have made considerable progress. Traditional luminescence probes include fluorescent derivatizing reagents, fluorescent probes and chemiluminescence probes which continue to develop. Of them, near infrared (NIR) fluorescent probes are especially suitable for the determination of biomolecules including proteins, so their development has been rapid. Novel luminescence probes (such as nanoparticle probes and molecular beacons) and resonance light scattering probes recently appeared in the literature. Preliminary results indicate that they possess great potential for ultrasensitive protein detection. This review summarizes recent developments of the above-mentioned probes for proteins and 195 references are cited.     

Transport proteins of Plasmodium falciparum: defining the limits of metabolism

In this review we give an account of transport processes occurring at the membrane interface that separates the asexual stage of Plasmodium falciparum from its host, the infected erythrocyte, and also describe proteins whose activities may be important at this location. We explain the potential clinical value of such studies in the light of the current spread of parasite resistance to conventional antimalarial strategies. We discuss the uptake of substrates critical to the survival of the intracellular malaria parasite, and also the parasite's homeostatic and disposal mechanisms. The use of the Xenopus laevis expression system in the characterisation of a hexose transporter ("PfHT1") and a Ca(2+) ATPase ("PfATP4") of the parasite plasma membrane are described in detail.     

Two-chamber AFM: probing membrane proteins separating two aqueous compartments

Biological membranes compartmentalize and define physical borders of cells. They are crowded with membrane proteins that fulfill diverse crucial functions. About one-third of all genes in organisms code for, and the majority of drugs target, membrane proteins. To combine structure and function analysis of membrane proteins, we designed a two-chamber atomic force microscopy (AFM) setup that allows investigation of membranes spanned over nanowells, therefore separating two aqueous chambers. We imaged nonsupported surface layers (S layers) of Corynebacterium glutamicum at sufficient resolution to delineate a 15 A-wide protein pore. We probed the elastic and yield moduli of nonsupported membranes, giving access to the lateral interaction energy between proteins. We combined AFM and fluorescence microscopy to demonstrate the functionality of proteins in the setup by documenting proton pumping by Halobacterium salinarium purple membranes.     

Pushing the limits for amplifying BrdU-labeled DNA encoding 16S rRNA: DNA polymerase as the determining factor

Identifying microorganisms that are active under specific conditions in ecosystems is a challenge in microbial ecology. Recently, the bromodeoxyuridine (BrdU) technique was developed to label actively growing cells. BrdU, a thymidine analog, is incorporated into newly synthesized DNA, and the BrdU-labeled DNA is then isolated from total extractable DNA by immunocapture using a BrdU-specific antibody. Analyzing the BrdU-labeled DNA allows for assessing the actively growing community, which can then be compared to the unlabeled DNA that represents the total community. However, applying the BrdU approach to study soils has been problematic due to low DNA amounts and soil contaminants. To address these challenges, we developed a protocol, optimizing specificity and reproducibility, to amplify BrdU-labeled gene fragments encoding 16S rRNA. We found that the determining factor was the DNA polymerase: among the 13 different polymerases we tested, only 3 provided adequate yields with minimal contamination, and only two of those three produced similar amplification patterns of community DNA.     

Pushing the detection limits: the evanescent field in surface plasmon resonance and analyte-induced folding observation of long human telomeric repeats

Conventional analysis of molecular interactions by surface plasmon resonance is achieved by the observation of optical density changes due to analyte binding to the ligand on the surface. Low molecular weight interaction partners are normally not detected. However, if a macromolecule such as DNA can extend beyond the evanescent field and analyte interaction results in a large-scale contraction, then the refractive index changes due to the increasing amount of macromolecules close to the surface. In our proof-of-principle experiment we could observe the direct folding of long, human telomeric repeats induced by the small analyte potassium using surface plasmon resonance spectroscopy. This work demonstrates the feasibility of new evanescent field-based biosensors that can specifically observe small molecule interactions.     

Invited review: probing the structures of muscle regulatory proteins using small-angle solution scattering

Small-angle X-ray and neutron scattering with contrast variation have made important contributions in advancing our understanding of muscle regulatory protein structures in the context of the dynamic molecular processes governing muscle action. The contributions of the scattering investigations have depended upon the results of key crystallographic, NMR, and electron microscopy experiments that have provided detailed structural information that has aided in the interpretation of the scattering data. This review will cover the advances made using small-angle scattering techniques, in combination with the results from these complementary techniques, in probing the structures of troponin and myosin binding protein C. A focus of the troponin work has been to understand the isoform differences between the skeletal and cardiac isoforms of this major calcium receptor in muscle. In the case of myosin binding protein C, significant data are accumulating, indicating that this protein may act to modulate the primary calcium signals from troponin, and interest in its biological role has grown because of linkages between gene mutations in the cardiac isoform and serious heart disease.     

Development of fluorescent biosensors for probing the function of motor proteins

Biosensors are becoming widely used both in basic research and in screening assays and reagentless sensors with fluorescent reporter groups attached to proteins form one class. This article describes the development of sensors for two small molecules, driven in particular by the need for high sensitivity and time resolution to probe mechanistic aspects of ATP-coupled motor proteins. The biosensors are for the products of the ATPase reaction, ADP and inorganic phosphate. The interplay between the possibilities for design and understanding the mechanism of the signal are discussed. Examples are described of how these sensors have been applied to understanding myosin and helicase motors.     

Use of bilirubin oxidase for probing chromophore topography in tetrapyrrole proteins

Bilirubin oxidase has been used to probe the surface topography of phycocyanins (C-phycocyanin and phycocyanin-645), peridinin-chlorophyll a-protein and phytochrome. The enzyme catalyzes oxidation of the tetrapyrrolic chromophores in these proteins. Relative rates of oxidation were 78.0 X 10(-6) s-1 (monitored at 617 nm) and 58.0 X 10(-6) s-1 (592 nm) for C-phycocyanin, 43.0 X 10(-6) s-1 for phycocyanin-645, 0.3 X 10(-6) s-1 (at 671 nm) and 1.3 X 10(-6) s-1 (at 480 nm) for peridinin-chlorophyll a-protein. The relative rate of free chlorophyllin a was 2.8 X 10(4) s-1 whereas upon binding to human serum albumin its rate of oxidation was reduced to 3.3 X 10(-3) s-1. Relative rates for the oxidation of Pr and Pfr forms of phytochrome were 2.9 and 19.5 s-1, respectively, which are consistent with earlier finding [( 1984) Plant Physiol. 74, 755-758] that indicated a preferential exposure of tetrapyrrolic chromophore in the Pfr form. In general, kcat/Km values derived from the Lineweaver-Burk plots followed the same trend as the relative rates of oxidation. For example, the kcat/Km for the free chlorophyllin a was 2.8 X 10(6) M-1 s-1 but it was only 1.1 M-1s-1 for the chlorophyll a in peridinin-chlorophyll a-protein where the chlorophyll is shielded by protein. These results reflect varying degrees of protection of the tetrapyrrolic chromophores from the enzymatic oxidation and prove that bilirubin oxidase can be generally used as a probe for deducing the topography of tetrapyrrolic chromophores.     

Simulating proton translocations in proteins: probing proton transfer pathways in the Rhodobacter sphaeroides reaction center.

A general method for simulating proton translocations in proteins and for exploring the role of different proton transfer pathways is developed and examined. The method evaluates the rate constants for proton transfer processes using the energetics of the relevant proton configurations. The energies (DeltaG((m))) of the different protonation states are evaluated in two steps. First, the semimicroscopic version of the protein dipole Langevin dipole (PDLD/S) method is used to evaluate the intrinsic energy of bringing the protons to their protein sites, when the charges of all protein ionized residues are set to zero. Second, the interactions between the charged groups are evaluated by using a Coulomb's Law with an effective dielectric constant. This approach, which was introduced in an earlier study by one of the authors of the current report, allows for a very fast determination of any DeltaG((m)) and for practical evaluation of the time-dependent proton population: That is, the rate constants for proton transfer processes are evaluated by using the corresponding DeltaG((m)) values and a Marcus type relationship. These rate constants are then used to construct a master equation, the integration of which by a fourth-order Runge-Kutta method yields the proton population as a function of time. The integration evaluates, 'on the fly,' the changes of the rate constants as a result of the time-dependent changes in charge-charge interaction, and this feature benefits from the fast determination of DeltaG((m)). The method is demonstrated in a preliminary study of proton translocation processes in the reaction center of Rhodobacter sphaeroides. It is found that proton transfer across water chains involves significant activation barriers and that ionized protein residues probably are involved in the proton transfer pathways. The potential of the present method in analyzing mutation experiments is discussed briefly and illustrated. The present study also examines different views of the nature of proton translocations in proteins. It is shown that such processes are controlled mainly by the electrostatic interaction between the proton site and its surroundings rather than by the local bond rearrangements of water molecules that are involved in the proton pathways. Thus, the overall rate of proton transport frequently is controlled by the highest barrier along the conduction pathway. Proteins 1999;36:484-500.     

Upper temperature limits of tropical marine ectotherms: global warming implications

Animal physiology, ecology and evolution are affected by temperature and it is expected that community structure will be strongly influenced by global warming. This is particularly relevant in the tropics, where organisms are already living close to their upper temperature limits and hence are highly vulnerable to rising temperature. Here we present data on upper temperature limits of 34 tropical marine ectotherm species from seven phyla living in intertidal and subtidal habitats. Short term thermal tolerances and vertical distributions were correlated, i.e., upper shore animals have higher thermal tolerance than lower shore and subtidal animals; however, animals, despite their respective tidal height, were susceptible to the same temperature in the long term. When temperatures were raised by 1°C hour(-1), the upper lethal temperature range of intertidal ectotherms was 41-52°C, but this range was narrower and reduced to 37-41°C in subtidal animals. The rate of temperature change, however, affected intertidal and subtidal animals differently. In chronic heating experiments when temperature was raised weekly or monthly instead of every hour, upper temperature limits of subtidal species decreased from 40°C to 35.4°C, while the decrease was more than 10°C in high shore organisms. Hence in the long term, activity and survival of tropical marine organisms could be compromised just 2-3°C above present seawater temperatures. Differences between animals from environments that experience different levels of temperature variability suggest that the physiological mechanisms underlying thermal sensitivity may vary at different rates of warming.