A questionnaire technique for assessing the stress at work

Subjective perception of the stress at work is a phenomenon reflecting the experience-mediated relationship between the sum of objective conditions of work and the sum of individual's traits available to cope with demands of the work environment. This relationship is mirrored in the function of the whole organism and can be described using the formula S = f (E, D), where S = the state of the organism, E = exposure (i.e. the sum of all stressors the organism is exposed to), and D = disposition (i.e. the sum of traits the organism has available to cope with these stressors). Based on these theoretical considerations, a questionnaire has been developed as a tool for assessing the work stress in different groups of occupations. This "Work Stress Assessment" (WSA) questionnaire was used in a group of 4800 female employees from various branches of the printing industry. Of a total of 160 questionnaire items, 98 items were selected and used to assess the workload in these employees, using a 5-point rating scale of evaluation. The responses were processed statistically, separately for 3531 blue-collar and 1269 white-collar respondents. The use of the factor analysis method revealed that the two groups of jobs had 16 factors in common, 3 factors were specific for blue-collar and 2 factors for white-collar jobs. Judged by factor components, the responses of subjects were well structured and the perceived stress at work turned out to well reflect the reality of conditions of work, which confirms the construct validity of this methodical approach based on the subjective perception of occupational stress. High coefficients of consistency (0.959 for blue-collar and 0.946 for white-collar occupations) point to the reliability of this questionnaire technique. Thus, the WSA questionnaire appears to suitably complement the other methodical approaches aimed at assessing the stress at work in different groups of occupations.     

Giant-cell carcinoma of the lung. A cytologic study

A case of giant-cell carcinoma of the lung, confirmed at autopsy, is presented. The cytologic features seen in sputum samples, bronchial washings and brushings and fine needle aspiration biopsy material as well as the histologic findings are described. The possible relationship to bronchioloalveolar carcinoma is discussed. The cytologic features of giant-cell carcinoma of the lung, when seen in the context of the clinical and radiologic setting, should allow the cytologic identification of the tumor prior to surgical intervention.     

A quantitative immunochemical technique for evaluation of the extent of integration of membrane proteins and of protein conformational changes and homologies

A methodology that allows an estimation of the number of antigenic determinants that remain exposed or accessible on a protein antigen after conformational changes, evolution, or integration in a membrane matrix has been developed. We have observed that a protein antigen, used as an immunogen, will stimulate the production of antibodies directed specifically against the exposed antigenic determinants of its surface, and then the maximum number of antibody molecules that can simultaneously bind to this antigen corresponds to the total covering of its surface. Surface modifications are shown to occur on Escherichia coli alkaline phosphatase upon noncovalent binding of inorganic phosphate and upon Zn²⁺ removal. The homology between pig intestinal and kidney brush border membrane aminopeptidases has been studied. When probed with anti-intestinal aminopeptidase (free form) the surfaces of both enzymes have been shown to bear six identical antigenic determinants, two of which are located in the area that is masked upon integration into the membranes. A technique to determine precisely the number of determinants rendered inaccessible by integration of aminopeptidases into the membranes is described. Two determinants out of the 12 of the “free intestinal aminopeptidase” surface are masked by integration in the membrane matrix. Thus, the methodology presented should be a valuable tool in all processes of molecular biology that involve protein surface probing.     

A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity

Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics.     

Improved transmission electron microscopy technique for the study of cytologic material

A modification of the technique of Coleman et al for the preparation of single cells in cytologic specimens for electron microscopy (EM) is described. By employing materials in the initial cytologic processing that are useful for EM, such as a paraformaldehyde-glutaraldehyde fixative, lactated Ringer's solution as a rinsing medium, glycerol as a mounting medium and cacodylate buffer for removal of coverslips, the use of alcohol fixatives and standard mounting media could be avoided. This preserved the cytoplasmic detail, which is usually degenerated in cells removed from cytologic specimens and processed for EM.     

A combined histochemical-neurophysiological technique for investigating the extent of diffusion of intracerebrally injected drugs

In order to 'dissect out' the function of a given region of the brain, the local application of a chemical transmitter probably parallels closely what is actually occurring biologically at the local site. This paper addresses itself to the problem of diffusion of drugs and describes the application of a modern device (the osmotic minipump) which produces a constant flow of minute quantities of the agent into the brain structure. Neurophysiological, behavioural, and histochemical investigations have been carried out in different species. Although the histochemical results of the minipump experiments indicate some spread, yet this seems to be at a concentration which would have no apparent pharmacological action. This was confirmed by our neurophysiological and behavioural studies and also by some recent reports in the literature.     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

Climatic mean diagrams: A technique for assessing the climatic differentiation of species

 A new method for comparisons of the ecogeographical and climatic constitutions of taxa is presented. The method will be important for understanding the evolution of species in time and space because ecogeographical characters of the species become directly comparable. On a global analytical level, distribution ranges of species may be considered as a function of the ecological constitution of the species, i.e. their endogenous hardiness to environmental forces, and climate. Frequency diagrams of the species' occurrences are calculated for the monthly climatic means of temperature and precipitation using high-resolution maps of distribution and climate. These frequency diagrams are used to construct monthly temperature-precipitation diagrams (TPD) that show the monthly climate spaces of the species'. For the first time, within the TPDs the climate spaces of the plants become directly comparable. However, comparability is somewhat restricted because of the necessity to consider the TPDs of all 12 months. A higher degree of abstraction is obtained using the climatic centre of a species as inferred from the TPD. The twelve monthly climatic means are transferred to the climatic mean diagram (CMD). In the CMD the climate spaces of the species become directly comparable in the course of the year. Despite the high degree of abstraction each diagram type reflects particular ecogeographical characteristics of the species even at a regional geographical level. Especially the CMD offers ways for understanding evolutionary shifts in the ecogeographical constitution of closely related species. The reasons for the shifts are composed of a hydric and thermal component. They may be addressed in molecular and physiological studies concerning evolutionary changes of these ecogeographical traits. Received August 7, 2000 Accepted November 5, 2001     

New criterial for cytologic diagnosis of adenoid cystic carcinoma

OBJECTIVE: To formulate new criteria for adenoid cystic carcinoma (ADCC). STUDY DESIGN: The usefulness of 17 items for a cytologically definitive diagnosis of ADCC was examined. The frequency (- - +++) of the 17 items in 18 cases of ADCC and 10 non-ADCC cases (pleomorphic adenoma, basal cell adenoma, myoepithelioma and epithelial-myoepithelial carcinoma) that displayed mimicking cytology was examined cytologically. The total score for cases of ADCC was compared with that for non-ADCC cases. RESULTS: The 17 items included broad intercellular spaces with adhesion; green, granular/cobwebby cytoplasm with translucent intercellular matrix with Papanicolaou staining; coarse hyperchromatin with little nuclear clearing; indistinct and partially distinct nucleoli; small nuclei; broad, smooth margin (SM) space; and translucent M/HG/SM. All cases with a total score of > 21 were ADCC (93.8%). All cases with a total score of < 12 were non-ADCC (87.5%). CONCLUSION: The 17 items appear to be useful as new criteria for ADCC.     

A study on enzymatic reaction using a liquid emulsion membrane technique

An enzymatic reaction using a liquid emulsion membrane technique was studied to investigate the effects of some experimental variables on the stability of liquid membrane, enzyme deactivation, and transport of substrates and products. The hydrolysis of L-phenylalanine methyl ester by alpha-chymotrypsin was selected as a model reaction system. First, a transport mechanism for the substrates and products across the membrane was qualitatively identified. Second, it was found that the pH of the internal phase was one of the most important variables to determine the enzyme activity in a liquid membrane. Third, the effect of membrane phase which consists of surfactant, carrier, and organic solvent on the emulsion stability was investigated. It was found that the properties of the organic solvents greatly affect the emulsion stability. For an optimum condition, it was possible to reuse the emulsion which consists of membrane phase and internal phase without further separation. It was finally concluded that the enzyme in a liquid membrane retained 60% of its native activity in spite of vigorous mixing during the emulsification step.     

Validation of merocyanine 540 staining as a technique for assessing capacitation-related membrane destabilization of fresh dog sperm

The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that had undergone membrane stabilization known to be associated with capacitation in other species, and whether such destabilization is detected earlier than when using the tyrosine phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence microscopy. Semen from nine dogs was collected and incubated in parallel in bicarbonate-free modified Tyrode's medium (−BIC), medium containing 15 mM bicarbonate (+BIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6 hours. Staining with M540-YP allowed the classification of dog sperm as viable without destabilized membranes, viable with destabilized membranes, nonviable without destabilized membranes, or nonviable with destabilized membranes. The percentage of viable sperm detected using EH (83.5 ± 1.37%; mean ± SEM) was higher than when using YP (66.7 ± 1.37%: P < 0.05; n = 54 semen samples). On the other hand, M540-YP identified a higher percentage of viable sperm with destabilized membranes than TP-EH (75 ± 1.76% vs. 35 ± 1.70%: P < 0.05; n = 54 semen samples). Staining with M540-YP indicated a rapid increase in the percentage of viable sperm with destabilized membranes, reaching a maximum during the first 30 minutes of incubation in +BIC. For all other treatments (i.e., −BIC, prostatic fluid, and PBS), the peak in the percentage of viable sperm with destabilized membranes was reached as much as 90 to 210 minutes later than incubation in +BIC. The lowest percentage of viable sperm showing signs of capacitation was recorded during incubation in PBS. We conclude that YP identifies sperm committed to cell death earlier than EH, and that the M540-YP stain combination identifies membrane destabilization known to be associated with capacitation in other species earlier than the TP-EH stain combination.     

A reliable method for assessing topographical arrangement of proteins in the chromaffin granule membrane

An efficient and reliable method for preparing chromaffin granules and membranes for topographical analysis has been designed to meet the following criteria. Only labeling of truly intact organelles is analyzed for cytoplasmic surface probes. No. resealing of open membranes occurs under specified reaction conditions. Saturation of available sites must be demonstrated. Problems in meeting these conditions are discussed, in particular the interference of granule lysate with labeling reactions.This work was supported by a grant from the National Institutes of Health, NS-13201.     

Suction sampling technique for obtaining standardized cytologic specimens from the oral mucosa

To enhance the value of exfoliative cytology for the study of the oral mucosa, a simple apparatus was developed to permit adequate sampling of a specific site so that samples collected on different occasions could be compared. The device essentially consists of a collecting cup connected to a blood collection evacuation system. The collecting cup is a modified female half of a stainless-steel filter holder supporting a 13-mm-diameter cellulose filter of 0.05-microns pore size. Suction pressure is applied by means of a 10-mL glass tube of premeasured vacuum. After positioning the collecting cup on the selected site on the buccal mucosa, the vacuum (440 mm Hg) is applied for five seconds. The mucosa is drawn in against the filter, producing a monolayered imprint of cells. This sample may be disengaged from the filter by agitation into a solution; this allows quantitative cytologic studies, such as the measurement of cell numbers by an electronic counter or the estimation of the areas of cells and nuclei by computer-aided image analysis of Cytospin preparations. Five separate samplings from each of three test subjects produced a harvest of 3,000 to 7,000 epithelial cells per sample; the cellular areas ranged from 784 to 1,052 sq microns while the nuclear areas ranged from 18.4 to 21.8 sq microns.     

A membrane technique for producing protoplasts ofCryphonectria parasitica

A method for the formation and regeneration of protoplasts of several strains of the chestnut blight fungus,Cryphonectria parasitica, is presented. The procedure utillizes cellophane membranes for growth and employs centrifugation for separation of protoplasts from hyphal fragments. Yields averaged 8.04×10⁶ protoplasts per membrane. Regeneration frequencies were 40–50% with a soft-agar overlay. These protoplasts are suitable for use in experiments designed to determine the role of dsRNA in hypovirulence ofC. parasitica.     

A membrane adsorption-SEM technique for observing neuston organisms

A technique to observe neuston organisms, combining membrane adsorption and scanning electron microscopy, is described. Bacteria, algae, and protozoa collected from the surface of 2 freshwater ponds using this methodology appeared well preserved by the fixation and dehydration procedures used.     

A membrane filter direct count technique for enumeratingBdellovibrio

A membrane filter direct count method was devised for enumeratingBdellovibrio cells in “clean” suspensions. The procedure involves filtering a specified volume of a diluted, Trisbuffered suspension ofBdellovibrio cells through a known area of a 100-nm-pore-size Millipore brand membrane filter. A clarification solvent was used to render the filter transparent, so that the bdeloyvibrios on the filter could be photomicrographed and counted either visually or by means of a Quantimet 720 Image Analyzing Computer. The number ofBdellovibrio cells per milliliter in the undituted suspension could be calculated from the mean number of cells per unit area of the filter, the dilution factor, and the volume of diluted suspension filtered. TheBdellovibrio cells were distributed on the filters in a Poisson manner when there were not more than about 3.5 cells per 100 μm² of filter surface. The membrane filter direct counts correlated well with direct counts obtained by the Petroff-Hausser method. The correlation of direct counts with plaque (“viable”) counts showed that 80 to 95% of the direct-countedBdellovibrio cells in the clean suspensions were capable of forming plaques on lawns of suitable substrate bacteria. *** DIRECT SUPPORT *** A01R4002 00007