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We have attempted to summarize current concepts pertaining to the ultrastructural evaluation of epithelial pulmonary neoplasms, with an emphasis in potential clinicopathological correlations. Routine ultrastructural evaluation of the most commonly encountered pulmonary carcinomas seems to provide, in a significant number of cases, additional information that may be of help in patient management. Immunocytochemistry when employed as an adjunct to electron microscopic evaluation of pulmonary tumors also plays an important role in clarifying morphologic findings. With the advent of specific chemotherapy protocols and future development of monoclonal antibodies in the treatment of neoplasms, accurate tumor classification becomes crucial. The combined use of electron microscopy and immunocytochemistry seems to be extremely useful in providing accurate and reproducible criteria of great potential value in the overall assessment of pulmonary neoplasms. In part II of this study we will address the importance of the ultrastructural evaluation of less common epithelial and mesenchymal pulmonary neoplasms.
Immunization of mice with a plasma membrane-enriched fraction from human malignant melanoma cells and subsequent generation of hybridomas resulted in the isolation of an IgG1 monoclonal antibody, 155.8, that recognizes chondroitin sulfate proteoglycans. By cell binding analysis, 155.8 was shown to react with seven of eight cultured melanoma cell lines, but not with a variety of lymphoblastoid cell lines or cultured tumor cells derived from other solid tumor types. Indirect immunoprecipitation of the 155.8 antigen from intrinsically labeled melanoma cells revealed a glycoprotein of Mr = 250,000 and a sulfated molecule of Mr greater than 400,000. The antigen was identified as a chondroitin sulfate type A/C proteoglycan synthesized by melanoma cells on the basis of its sensitivity to chondroitinase ABC digestion and the identification of sulfated glycosaminoglycans released from the antigen immunoprecipitated by 155.8. The determinants recognized by antibodies 155.8 and 9.2.27, another anti-chondroitin sulfate proteoglycan, immunoprecipitate only a proteoglycan from high density cesium chloride gradient fractions, (1.487 g/liter); however, they immunoprecipitate a free glycoprotein of Mr = 250,000 from low density fractions (1.317 g/liter). This demonstrated that the 155.8 and 9.2.27 determinants, both of which reside on the glycoprotein of Mr = 250,000, are also present in the proteoglycan, suggesting that this glycoprotein is the proteoglycan core protein. Monoclonal antibody 155.8 reacts with a determinant on the core protein distinct from that recognized by 9.2.27. Proteoglycans bearing 155.8 determinants are distributed on the surface of cultured melanoma cells in a punctated fashion that apparently resolves to short, filamentous structures at high magnification. Immunohistochemical analysis demonstrated that 155.8-defined proteoglycans are found in freshly biopsied melanoma tissue, suggesting that these antigens are also synthesized in vivo by melanoma cells.
Small amounts of a 22,000-dalton (22Kd) Ia polypeptide were immunoprecipitated with monoclonal anti-Ia antibodies from murine B lymphocyte tumor cells and from splenocytes that express the I-Ad subregion. HPLC peptide map comparisons of A alpha d, A beta d and the 22Kd polypeptides demonstrated that the 22Kd polypeptide has extensive homology with A alpha d. Amino terminal sequence analysis of the 3H-tyrosine 22Kd polypeptide indicates this polypeptide has tyrosines at residues 16, 17, 30, and 32. Unexpectedly, this amino terminal 22Kd sequence is homologous with A beta d rather than A alpha d. These results suggest that the 22Kd polypeptide is a hybrid molecule containing both A alpha d and A beta d-like sequences. The 22Kd polypeptide has provisionally been designated A delta d.
The fine purification of an alkaline protease (thermitase) from Thermoactinomyces vulgaris by means of isoelectrical focussing in the flat-bed procedure using granulated gel is reported. An Na2SO4-precipitated crude product serves as the starting material. Isoelectrical focussing leads in a single step to a highly purified protein with an uniform N-terminal end group. The enzyme has an IP at 9.0 and a mol. wt. of 37,400; it consists of a polypeptide chain with arginine as the N-terminal, and tyrosine as the C-terminal end group. In addition to an essential serine residue, a SH group could be demonstrated which is hardly accessible in the native enzyme. Furthermore, the influence of different protease inhibitors was studied.
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Campylobacter coli CK 205, isolated from swine feces, was examined for changes in cell morphology, protein and lipooligosaccharide (LOS) patterns during starvation-survival experiments. Bottles filled with sterile filtered A. dest. were seeded with campylobacters and incubated at 4 degrees C and 37 degrees C. Transition to the nonculturable stage occurred within 48 hours (37 degrees C) and 2 weeks (4 degrees C), respectively. In contrast to the culturability the electrophoretic studies showed no changes in whole cell protein or LOS patterns. The electron microscopic pictures revealed spiral and coccoid forms, partly with a slightly enlarged periplasmatic space or budding of the membrane. Totally intact non culturable spiral or coccoid forms might be regarded as dormancy forms that cannot be detected by conventional microbiological methods in water examination.
The effects on the channel characteristics of four single amino acid substitutions in OmpF porin and of a deletion mutant in the constriction loop L3 have been studied. These mutations are all located in the narrow section of the channel of the protein that forms pores across the outer membrane of Escherichia coli. The single channel conductance of the deletion mutant (Delta109-114) is decreased by one third, whereas the point mutations do not exhibit significant deviations from that of the wild-type protein. The mutants exhibit drastic changes in ion selectivities. In the wild-type protein, the critical threshold potential (Vc), above which channels close reversibly, exhibits a strong pH dependence, with a titration point of approximately pH 7.7, which is abolished in all mutants studied here. Diffusion of six monosaccharides is little affected in the point mutants, while four disaccharides are taken up at highly increased rates by the deletion mutant. The functional results, presented here, are correlated to the x-ray structures of the mutants (Lou, K.-L., Saint, N., Prilipov, A., Rummel, G., Benson, S.A., Rosenbusch, J.P., and Schirmer, T. (1996) J. Biol. Chem. 271, 20669-20675). In most, but not all, cases, the structural changes explain the functional alterations observed.
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The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.
BACKGROUND AND STUDY AIMS: Although it is time-consuming to measure the volume of lesions using three-dimensional endoscopic ultrasonography (3D-EUS), the technique is suitable for tissue characterization, as it allows images of multiple areas to be obtained simultaneously in uniform conditions. The present study tested automatic volume measurement using tissue characterization based on 3D-EUS. MATERIALS AND METHODS: Nine polygonal sections of resected spleen (volume 0.66 +/- 0.19 cm3) were immersed in water, and 40 radial 3D-EUS images were obtained. For tissue characterization, the methods of co-occurrence matrix and gray-level difference and discriminant analysis were used. Each spleen section was also measured using 3D-EUS. The volume of tissue identified as spleen using tissue characterization and the volume calculated on the basis of the 3D-EUS images were both compared with the actual volume measured beforehand. Measurements using tissue characterization and 3D-EUS were carried out for every third image. In three clinical cases of cancer the volume of the lesion was measured using tissue characterization and 3D-EUS. RESULTS: The mean volume of the nine splenic sections estimated using tissue characterization was 1.2 +/- 0.41 cm3 (mean +/- SD), while the mean volume estimated with 3D-EUS imaging was 1.1 +/- 0.30 cm3 (mean +/- SD). The volumes measured using tissue characterization were on average 13% larger than those obtained with 3D-EUS. Linear regression analysis showed a high degree of correlation between the two sets of measurements (r=0.97, P<0.00005), and also showed a high correlation between the volumes obtained using tissue characterization and the actual volume (r=0.93, P<0.0005). However, the volumes calculated using 3D-EUS images were larger than the actual volume (61% on average), and the volumes estimated using tissue characterization were also greater than the actual volume. The overestimation reflected the fact that measurement was only carried out in every third 3D-EUS image. In the clinical cases, the mean value for "true" tumor tissue as determined on EUS imaging represented 73% of the volume interpreted as cancer using tissue characterization. CONCLUSIONS: There was a good correlation between the volume measured with 3D-EUS and the volume obtained using tissue characterization. The tissue characterization volumes were only relatively slightly larger than the volumes measured using 3D-EUS, suggesting that there may be some promise for this application of tissue characterization.
The U.S. Department of Energy has selected three sites, from five nominated, to characterize for a nuclear repository to permanently dispose of nuclear waste. This decision was made without the benefit of an analysis of this "portfolio" problem. This paper analyzes different portfolios of three sites for simultaneous characterization and strategies for sequential characterization. Characterization of each site, which involves significant subsurface excavation, is now estimated to cost $1 billion. Mainly because of the high characterization costs, sequential characterization strategies are identified which are the equivalent of $1.7-2.0 billion less expensive than the selected DOE simultaneous characterization of the three sites. If three sites are simultaneously characterized, one portfolio is estimated to be the equivalent of $100-400 million better than the selected DOE portfolio. Because of these potential savings and several other complicating factors that may influence the relative desirability of characterization strategies, a thorough analysis of characterization strategies that addresses the likelihood of finding disqualifying conditions during site characterization, uncertainties, and dependencies in forecast site repository costs, preclosure and postclosure health and safety impacts, potential delays of both sequential and simultaneous characterization strategies, and the environmental, socioeconomic, and health and safety impacts of characterization activities is recommended.
In the field of risk assessment, characterizing the nature and magnitude of human health or environmental risks is arguably the most important step in the analytical process. In this step, data on the dose-response relationship of an agent are integrated with estimates of the degree of exposure in a population to characterize the likelihood and severity of risk. Although the purpose of risk characterizations is to make sense of the available data and describe what they mean to a broad audience, this step is often given insufficient attention in health risk evaluations. Too often, characterizations fail to interpret or summarize risk information in a meaningful way, or they present single numerical estimates of risk without an adequate discussion of the uncertainties inherent in key exposure parameters or the dose-response assessment, model assumptions, or analytical limitations. Consequently, many users of risk information have misinterpreted the findings of a risk assessment or have false impressions about the degree of accuracy (or the confidence of the scientist) in reported risk estimates. In this article we collected and integrated the published literature on conducting and reporting risk characterizations to provide a broad, yet comprehensive, analysis of the risk characterization process as practiced in the United States and some other countries. Specifically, the following eight topics are addressed: (1) objective of risk characterization, (2) guidance documents on risk characterization, (3) key components of risk characterizations, (4) toxicity criteria for evaluating health risks, (5) descriptors used to characterize health risks, (6) methods for quantifying human health risks, (7) key uncertainties in risk characterizations, and (8) the risk decision-making process. A brief discussion is also provided on international aspects of risk characterization. A number of examples are presented that illustrate key concepts, and citations are provided for approximately 100 of the most relevant papers.
A variety of methods is now available for characterizing species and strains of Theileria. For many practical purposes involving field control of theileriosis, characterization on a broad basis may be sufficient, but in other areas much more precise characterization is required. Such precision can be usefully exploited only when cloned parasite populations are involved, and methods to improve parasite characterization and parasite cloning should be developed concurrently. The current methods of immunization against theileriosis involve the use of live parasite populations which are generally poorly defined and, in addition, have the capacity to undergo biological change (by selection, mutation or genetic recombination) within hosts and vectors. Such changes may be difficult to define and identify, but could have profound effects on immunization strategies. Improved methods of parasite characterization and selection, which are now becoming available, will enable parasite stocks for immunization to be identified and selected more precisely, and any biological changes that occur can be monitored. Improved methods of parasite characterization will also open the way to a better understanding of Theileria genetics and the mechanisms of heritability, which appear to differ in some fundamental ways from patterns of Mendelian inheritance. Controlled matings between selected and defined populations of parasites can be envisaged, with the aim of producing hybrid parasites for immunization. In addition, the prospects of modifying the theilerial genome by genetic manipulation become very real: transfection vectors tailored by restriction enzymes could be used to insert or modify gene sequences to develop parasites with appropriate sets of characters. It may also be possible to identify parasite genes which trigger the cytotoxic response which is so important in immunity (Eugui and Emery, 1981; Emery et al., 1981; Preston et al., 1983). Such genes might then be transfected into bovine host lymphocytes to generate immunity against the whole parasite (Iams, 1985). The gene products which are responsible for stimulating immune responses could also be synthesized artificially and used for vaccination. Methods of characterizing Theileria range from Giemsa's staining to DNA hybridization; all have a role to play, and by judicious selection of appropriate methods for particular circumstances, it is becoming possible to characterize theilerial parasites very precisely. Improved methods of characterization can, in turn, lead to a better understanding of parasite biology and to the development of improved methods of immunization and control.
Adverse effects of chemicals on humans are typically assessed following four steps: hazard identification, hazard characterization, exposure assessment and risk characterization. Hazard characterization is defined as the qualitative and/or quantitative evaluation of the nature of the adverse effects associated with biological, chemical and physical agents of interest. For chemical agents, hazard characterization is based on a series of in vitro and/or in vivo data obtained from mechanistic, kinetic and dose-response studies on the agent of concern, which are analyzed and integrated for extrapolation to eventually match human conditions. Thus, an accurate experimental design and the development of test methods capable of generating data relevant to hazard characterization are essential for the useful risk assessment of chemicals, including inhaled materials. It should, however, be stressed that hazard characterization has widely been limited to single chemicals. The hazard characterization of airborne mixtures therefore poses a new problem in toxicology, which calls for a novel approach to its scientific assessment. During the last three decades, a number of epidemiological and experimental studies have been conducted focusing on two kinds of inhaled complex mixtures, namely cigarette smoke and diesel exhausts. A new approach to the assessment of airborne complex mixtures may be elaborated through the appropriate, combined use of the findings of such studies. In this context, the present review article is intended to illustrate some basic strategies for and technical issues related to the hazard characterization of inhaled complex mixtures, thereby taking up representative epidemiological and experimental data from published papers on tobacco smoke.
Capillary isoelectric focusing (cIEF) can be a useful tool for the characterization and identification of microbes. Based on the whole column imaging detection (WCID) technique and using plant growth-promoting rhizobacteria (PGPR) as test microbes, we present a two-level cIEF characterization method for the characterization and identification of bacteria. Intact bacteria were first characterized according to their apparent isoelectric points measured by cIEF-WCID and then lysed bacteria were further characterized by cIEF profiling of the intracellular proteins. Cellular clustering was found to be the main experimental barrier for the characterization of intact bacteria. The addition of sodium chloride (100mM) to the sample mixture was found to be an effective way to reduce clustering. Due to the high efficiency and high resolution of cIEF-WCID, characterization of bacteria according to their intracellular proteins can be implemented simply and quickly without optimization of the experimental conditions. To improve the detection sensitivity with laser induced fluorescence (LIF)-WCID, the possibility to label bacteria with a non-covalent fluorescent dye, NanoOrange, was explored.
This review places the characterization of interactions by biosensor technology in the broader context of their study by quantitative affinity chromatography. The general reluctance to consider biosensor-based characterization as a form of quantitative affinity chromatography on the grounds of a difference in aims of the two techniques reflects a mistaken belief that BIAcore and IAsys studies characterize the kinetics of the chemical reaction responsible for biospecific adsorption of a soluble reactant to an immobilized form of its affinity partner. It now transpires that the association and dissociation rate constants thereby determined refer to thermodynamic characterization of biospecific adsorption in terms of a single-phase model in which affinity sites are distributed uniformly throughout the liquid-phase volume accessible to the partitioning reactant--the model used for characterization of biospecific adsorption by quantitative affinity chromatography. In that light the most important attribute of biosensor technology is its potential for thermodynamic characterization of biospecific adsorption by virtue of its ability to monitor complex formation directly; and hence its potential for the characterization of interactions with affinities that are too strong for study by forms of quantitative affinity chromatography that monitor complex formation on the basis of reactant depletion from the liquid phase. Kinetic as well as thermodynamic analyses of biosensor data are described for attainment of that potential.