Suitable Labels Research Articles

The measurement of tissue protein turnover

Tissue protein turnover can be assessed by a number of semi-, quantitative and qualitative methods. There are a number of static indices of the state of turnover of protein, for example amount of RNA per DNA or protein, the state of aggregation of ribosomes (i.e. the polyribosome index), the abundance of mRNA for particular proteins, and the enzymatic activity of proteins such as proteases, ribonuclease, etc. In addition, the concentration of particular amino acids such as glutamine or non-re-utilizable amino acids, formed post-translationally, such as 3-methylhistidine or hydroxyproline, are able to provide snapshot indices. However, since turnover is a dynamic process it should, ideally, be probed using methods such as the incorporation of tracer amino acids into protein or the dilution of tracer amino acids in the free pool by protein breakdown. The combination of tracer and tissue or limb balance methods is especially powerful since all the dynamic processes can potentially be quantified. The use of stable isotopes to label metabolic tracers has dramatically increased the feasibility of carrying out measurements of protein synthesis and breakdown and there has been a substantial growth in the application of the methods to a wide variety of tissues sampled by biopsy or at operation. Summaries of a number of currently feasible methods are provided, together with commentary on the relative efficacy of the methods and of the instrumental techniques required. There is also a discussion of suitable tracer labels and amino acids, plus a summary of the most reliable current values for protein turnover in a variety of tissues. The review also contains descriptions of potential methods which have not yet been applied in human beings but which are feasible, given the current recent increases in the accuracy and sensitivity of instrumentation for measurement of stable isotope labelling.

An enzymatic amplification cycle for high sensitive immunoassay

An amplification cycle consisting of oligosaccharide dehydrogenase (ODH) and laccase is found to be highly sensitive to several catecholamines and p-aminophenol. An immunoassay is constructed by combining this amplification cycle with a suitable enzyme label, of which p-aminophenol is the product of the enzymatic reaction. Alkaline phosphatase and β-galactosidase were tested as labels for immunoglobulin G from goat (gtIgG) and human thyroid stimulating hormone (hTSH) immunoassay. The combination of enzymatic amplification cycles with a sandwich type immunoassay yields high sensitivity measurement of hTSH. The assay was found to have two linear ranges: the first one is between 0.2 and 20 ng/ml, the second one from 0.0005 to 0.5 ng/ml, depending on assay procedure. Both together cover well the diagnostically relevant range of 0.1–100 mU/l (0.017-17 ng/ml); the detection limit is at 0.3 pg/ml (1.8 μU/l).

Ultrasensitive biosensors

Bi-enzymatic amplifiers using cyclic reactions consisting of NADH independent dehydrogenases and phenol oxidases were found to be highly sensitive to several catecholamines and p-aminophenol. Amplification factors range from several 100 to up to 10 000 depending on substrate and enzyme loading. The detection limit for catecholamines was subnanomolar. An electrode based on tyrosinase/glucose dehydrogenase responds to peptides containing tyrosine. Applications to monitoring of cell activities as well as to immunodetection are described. Immunoassays were constructed by combining the amplification cycle with a suitable enzyme label, of which p-aminophenol is the product of the enzymatic reaction. Alkaline phosphatase and β-galactosidase were tested as labels for immunoglobulin G from goat (gtIgG), as well as adaption to hapten determination in a displacement format.

The complexity of understanding line drawings of origami scenes

This paper deals with the interpretation of line drawings of Origami scenes (Kanade, 1980), that is scenes obtained by assembling planar panels of negligible thickness, and it addresses the computational complexity of the problem of consistently assigning suitable labels to the segments describing 3D properties as convexity, concavity and occlusion (labeling problem). The main results of the paper are the following: (a) the labeling problem for line drawings of Origami scenes is NP, as for the case of trihedral scenes; (b) the problem remains NP even if the location of the vanishing points in the image plane is given, whereas for trihedral scenes the problem was polynomially solvable; (c) in case the vanishing points are known the labeling problem can be subdivided into two subproblems, the paneling problem and the labeling-a-paneled-line-drawing problem which are both polynomially solvable (there may be, however, exponentially many legal panelings to check against labelability). The approach provides geometrical constraints which help select ‘natural’ interpretations even in the presence of occlusions and in the absence of apparent 3D-symmetries, and which may help highlight the role of geometrical regularities in the design of biologically plausible algorithms for the interpretation of line drawings.

Auto-Tuning of Low-Order Controllers by Direct Manipulation of Closed-Loop Time Domain Measures

Many controller tuners are based on linear models of both the controller and process. Desired performance is often predetermined or adjusted in a manner that is not directly related to the desired response. All physical processes contain non-linearities, commonly of the actuator saturating type, and many controllers contain heuristics for implementation in real systems, such as anti-integral wind-up in PID (proportional integral differential) controllers. For different processes a range of closed-loop response shapes are desired, often described by features of the response such as rise time, overshoot and settling time. This paper investigates the possibility of basing controller tuning on closed-loop system response data such that desired performance is incorporated directly in terms of familiar time domain features or labels, thus eliminating the need for a mathematical process model and repeated tuning reformulations to achieve the desired performance. A controller tuning method named label-based neuro-tuning (LBNT) is developed and analysed by application to PID controller tuning for process models indicative of real process behaviour. Simulations and numerical investigation indicate that LBNT is a viable technique for the tuning of low-order SISO (single-input-single- output) controllers. Tuning is straightforward, flexible and copes well with process parametric changes and performance specification reformulation. The drawbacks are a complicated pretune phase, a limited selection of suitable labels and a difficulty in defining general classes of tuning problems for its application. The technique is not based on the assumption of process linearity, but due to the inability to characterize classes of input signals and operating points the types of process non-linearity are restricted. The controller may be non-linear, but must be structurally predetermined, and an input/output process model of arbitrary structure is required.

High-level Biosynthetic Substitution of Methionine in Proteins by its Analogs 2-Aminohexanoic Acid, Selenomethionine, Telluromethionine and Ethionine in Escherichia coli

We have utilized a T7 polymerase/promoter system for the high-level incorporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high-level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high-level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray structural analysis, supplementing the traditional trial-and-error preparation of heavy-atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high-level incorporation of amino acid analogs can provide single-atom mutations for the detailed study of the structure and function of proteins.

High-level Biosynthetic Substitution of Methionine in Proteins by its Analogs 2-Aminohexanoic Acid, Selenomethionine, Telluromethionine and Ethionine in Escherichia coli

We have utilized a T7 polymerase/promoter system for the high-level incorporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high-level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high-level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray structural analysis, supplementing the traditional trial-and-error preparation of heavy-atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high-level incorporation of amino acid analogs can provide single-atom mutations for the detailed study of the structure and function of proteins.

Performance evaluation of coded modulation schemes based on binary lattices

We describe computational techniques for the evaluation of error probability of modulation schemes based on binary lattices and multilevel coding. These techniques stem from the representation of a binary lattice in the form of a trellis whose structure depends on the block codes on which the binary lattice is based. Multilevel coded-modulation schemes can also be described through a trellis; by combining with this the trellis structure of the lattice constellation we obtain a single trellis that can be used for maximum-likelihood decoding using the Viterbi algorithm and for computing the error probability. An upper bound to error probability can be obtained from the transfer function of the trellis with suitable labels, and is applicable to both maximum-likelihood and staged decoding. >

Covalent inhibitors of P-glycoprotein ATPase activity.

Verapamil-stimulated ATP hydrolysis by Chinese hamster P-glycoprotein in plasma membranes was shown to occur at a site(s) which is conformationally flexible and of relatively low affinity and specificity. Such properties distinguish P-glycoprotein from other transport ATPases. 8-Azido-ATP and 2-azido-ATP were excellent substrates, confirming that both analogs are suitable photoaffinity labels for investigating the catalytic site(s). Inactivation of ATPase activity occurred coincident with covalent incorporation of approximately two 8-azido-ATP/P-glycoprotein, with the incorporated analog distributed equally between N- and C-terminal halves of the molecule. N-Ethylmaleimide potently inactivated in an ATP-protected, dithiothreitol-irreversible manner, with maximal inactivation occurring coincident with incorporation of approximately two N-ethyl-maleimide/P-glycoprotein. The critical catalytic site sulfhydryls were shown to be located equally in N- and C-terminal halves of the molecule. Sulfhydryl-substituted purines also gave substantial inhibition of P-glycoprotein ATPase activity, which was dithiothreitol reversible. The data provide guidelines for beginning investigation of catalytic site architecture by protein chemistry approaches.

14C and125I Labelling of Humic Material for Use in Environmental Studies

Abstract Humic and fulvic acids are present in nearly all natural waters. These acids are known to affect the transport of environmental contaminants such as metals and hydrophobic organics through the terrestrial environment. an understanding of their role in the transport of contaminants is therefore essential and is facilitated if the acid is labelled with a suitable radioactive label. This paper reports the use of 14C-methylamine and 125I to label humic acid with either 14C or 125I and investigates factors which affect the yield of these reactions. the stability and mobility of the labelled humic material through sand is also reported.

Electrostatic characterization of liposomes

A variety of experimental techniques can be used to probe the electrostatic characteristics of lipid bilayers and liposomes. Many are based on the titration of the charge-dependent liposome properties. Determination of the lipid (headgroup) conformation, bilayer phase transition temperature, interbilayer force, mobility in external electric field, or transbilayer transport are but a few examples of this. Laser-Doppler velocimetry, based on dynamic light scattering by the lipid suspension, is probably the best currently available tool for the fast and/or routine determination of the electrostatic potential of liposomes; the proviso for this is that data analysis is done prudently. In addition to this potential-dependent adsorption or binding of ion or ionic probes to the liposome surface can be measured. Interesting organic ions can be made visible by suitable (fluorescent, nuclear, paramagnetic, etc.) labels. The conclusions drawn from any ion-liposome interaction study are only reliable, however, if Coulombic, as well as non-trivial electrostatic effects are both accounted for. When the magnitude of the latter is not known theoretically, the accuracy of the data analysis can be improved by subtracting a complementary set of data (measured with uncharged lipid vesicles or in a highly concentrated salt solution) from the corresponding results obtained with charged liposomes or at low salt concentration.

Luminescence of Europium(III) Chelates with 4‐(Arylethynyl)pyridines as Ligands

AbstractSome Spectral properties and luminescence intensities of EuIII chelates with 4‐(arylethynyl)pyridine‐2,6‐dicarboxylic acids 1–15 and 2,2′,2″,2′″‐{[4‐(arylethynyl)pyrridine‐2,6‐diyl]bis(methylenenitrilo)} tetrakis(acetic acids) 16–26 were measured both in H2O and EtOH solutions for the purpose of developing suitable labels to be used in time‐resolved luminescence‐based bioaffinity assays (Tables 1 and 2). The substitution at the Ar group has a significant effect upon the observed luminescence intensities, excitation wavelengths, and decay constants of the complexes, Moreover, the changes in the environment cause great variation in those properties of certain EuIII chelates.

New Heteroaromatic Complexing Agents and Luminescence of Their Europium(III) and Terbium(III) Chelates

AbstractTwelve heteroaromatic complexing agents 9a–I were synthesized with the purpose to develop suitable labels for time‐resolved luminescence‐based bioaffinity assays. The relative luminescence yields, excitation maxima, and emission decay constants of their europium(III) and terbium(III) chelates were determined. According to these results, 2,2′,2″,2‴‐[(2,2′‐bipyridine‐6,6′‐diyl)bis(methylenenitrilo)]tetrakis (acetic acid) (9e) and 2,2′,2″,2‴‐[(2,2′:6′,2″‐terpyridine‐6,6″‐diyl)bis(methylenenitrilo)] tetrakis(acetic acid) (91) are the most promising agents.

Synthesis and bioactivity of photoaffinity labels of the plant growth regulator 1-(3-chlorophthalimido)cyclohexanecarboxamide (AC 94377)

AC 94377 has been shown to mimic the growth-regulating activity of gibberellins. For the investigation of the receptor protein binding site associated with AC 94377 bioactivity, suitable photoaffinity labels are required. Therefore, five photoaffinity-labeled analogues of AC 94377 have been synthetisized: 1-(3-chlorophthalimido)cyclohexanecarboxazide (4), 1-(3-azidophthalimido)cyclohexanecarboxamide (8), 1-(3-azido-6-fluorophthalimido)cyclohexanecarboxamide (14,), 1-(4-azidophthalimido)cyclohexanecarboxamide (18), and 1-(3-azido-4-methylphthalimido)cyclohexanecarboxamide (24) (...)

Radioisotopic model for investigating thromboembolism in the rabbit

111Indium ( 111In)-oxine labeled platelets have been used in a variety of species to assess platelet behavior in vivo. We have shown that 111in-oxine is a suitable label for rabbit platelets and, using a noninvasive technique for the automated, continuous, external imaging of these radiolabeled platelets, we have shown that intravenous adenosine disphosphate (ADP), collagen, platelet activating factor (PAF), and thrombin all elicit dose-related accumulation of platelet-(but not erythrocyte-)associated radioactivity in the thoracic region and a concomitant fall in both the cranial and hindlimb regions of the anesthetized rabbit. Intracarotid (i.c.) ADP, collagen, PAF, and thrombin also produce dose-related increases in platelet-associated radioactivity in the thoracic and decreases in the cranial and hindlimb regions. However, the initial fall in cranial counts induced by i.c. thrombin was followed by a marked increase that was sustained for up to 3 hr. These results suggest this may be a useful model for investigating the mechanisms of platelet activation in the arterial and venous circulations in vivo and may provide a novel model for investigating thromboembolic events in the cerebral circulation.

Behavior of spin labels in a variety of interdigitated lipid bilayers

The behavior of a number of spin labels in several lipid bilayers, shown by X-ray diffraction to be interdigitated, has been compared in order to evaluate the ability of the spin label technique to detect and diagnose the structure of lipid bilayers. The main difference between interdigitated and non-interdigitated gel phase bilayers which can be exploited for determination of their structure using spin labels, is that the former have a much less steep fluidity gradient. Thus long chain spin labels with the nitroxide group near the terminal methyl of the chain, such as 16-doxylstearic acid, its methyl ester, or a phosphatidylglycerol spin label containing 16-doxylstearic acid (PG-SL), are more motionally restricted and/or ordered in the interdigitated bilayer than in the non-interdigitated bilayer. This difference is large enough to be of diagnostic value for all three spin labels in the interdigitated bilayers of dipalmitoylphosphatidycholine /glycerol, dipalmitoylphosphatidylglycerol/polymyxin B, and the asymmetric 1-stearoyl-2-caproylphosphatidylcholine. A difference can also be detected for all three spin labels in the interdigitated bilayers of dihexadecylphosphatidylcholine, dipalmitoylphosphatidylcholine/ethanol, and 1,3-dipalmitoylphosphatidylcholine. However, it is not large enough to be of diagnostic value at low temperatures. Use of probes with the nitroxide group closer to the apolar/polar interface reveals that these latter interdigitated bilayers are more disordered or less closely packed. As the temperature is increased, however, the motion of the PG-SL does not increase as much in these interdigitated bilayers as in non-interdigitated bilayers. The difference in the motion and/or order of PG-SL between interdigitated and non-interdigitated bilayers is large enough at higher temperatures to be of value in diagnosing the structure of the bilayers. Thus by choice of a suitable spin label and a suitable temperature, this technique should prove useful for detection and diagnosis of lipid bilayer structure with a good degree of reliability. Caution must, of course be exercised, as with my spectroscopic technique. Spin labels will also be invaluable for more detailed studies of known interdigitated bilayers, which would be time- and material-consuming, if carried out using X-ray diffraction solely.

Names for Aneurysms-Reply

In Reply.— Dr Yao raises the issue of the significance of the term aneurysm. We find the following in The Heart: Arteries and Veins 1 : It is important to recognize the distinct nature of this lesion. The term dissecting aneurysm is often applied but leads to confusion with an entirely separate problem—expanding or leaking aneurysm of atherosclerotic or luetic origin. Aortic dissection is a much more suitable label, particularly since aneurysm formation is not a feature of the acute phase. External rupture, by far the most common cause of death from aortic dissection, tends to occur at the site of the entrance tear. This validates my comments in the original book review that aortic dissections are primarily a disease of the thoracic aorta, while aneurysms are primarily a disease of the abdominal aorta. It is true that late aneurysm rupture may complicate thoracic aortic dissections; however, as noted above,

Monocytes become macrophages; they do not become microglia: a light and electron microscopic autoradiographic study using 125-iododeoxyuridine.

This light and electron microscopic autoradiographic study of stab injuries in the spinal cord of mice evaluated the ultrastructural characteristics of cells labeled by incorporation of the thymidine analogue 125I-5-iodo-2'-deoxyuridine (I-UdR), injected one day prior to injury. I-UdR was used instead of tritiated thymidine (H-TdR) because H-TdR can be reutilized and is therefore not a suitable pulse label for long-term studies of cell migration. Using serial thick and thin sections for autoradiography 614 labeled cells were identified. Labeled cells included 545 monocytes/macrophages, 50 lymphocytes, 17 pericytes, one endothelial cell, and one arachnoid cell. No labeled cell had the morphology of microglia. We concluded that macrophages in stab injuries of the spinal cord of mice are derived from blood monocytes. Blood-derived lymphocytes are also involved in the reaction to spinal cord stab injury. Microglia are not blood-derived and are not seen as a transitional form in the differentiation of monocytes to macrophages.

Studies directed toward labeling analysis of angiotensin II in plasma.

We assay a 1-mL plasma sample containing angiotensin II (103 pg by radioimmunoassay) for the hormone by the following sequence of steps: add 125I-labeled val5-angiotensin II as an internal standard, extract on a C18 Sep Pak column, extract on an antibody affinity column, label the extract with an 125I Bolton-Hunter reagent, separate on a Bio Gel P2 column, and repetitively separate on a reversed-phase "high-performance" liquid-chromatographic column, detecting the eluting compounds by counting radioactivity. The fact that we measured 46 pg of angiotensin II-like substance per milliliter in a sample of pooled plasma is encouraging for the further development of this methodology. In particular, replacing the radioisotope with a more suitable chemical label such as an electrophoric (electron-capturing) release tag should be useful.

A dual laser analysis of the migration of XRITC-labeled, FITC-labeled, and double-labeled lymphocytes in sheep.

Substituted rhodamine isothiocyanate (XRITC) has been used to study lymphocyte migration in sheep. After being labeled in vitro with XRITC, lymphocytes appeared in the efferent lymph of single lymph nodes with the same kinetics as cells labeled with fluorescein isothiocyanate (FITC). The recovery of intravenously injected XRITC-labeled cells was followed in lymph for several days. The kinetics and recoveries were compared with data obtained using FITC, chromium-51, and indium-111. XRITC was found to be a suitable label and, using dual laser (argon and krypton) flow cytometry, it could be analyzed simultaneously with FITC. In addition, it was possible to relabel FITC-stained cells with XRITC after they were recovered in lymph. The migratory characteristics of such double-labeled cells were not different from single-labeled cells.