Characterization of a CV-1 cell cycle. III. Biophysical parameters.

Membrane preparations from three independently selected concanavalin A-resistant cell lines incorporated si$\text{g}$nificantly less GDP-[¹⁴C]mannose into lipid, oligosaccharide-lipid and protein fractions than preparations obtained from parental wild populations. The results from experiments with membranes from a revertant concanavalin A-resistant line more closely resembled the wild-type populations. The amount of mannose label incorporated into glycoprotein in the variant cells was higher than expected if it is assumed that the pathway GDP-mannose → mannolipid → oligosaccharide-lipid → mannoprotein is functioning in these cells. Evidence is presented to suggest that conversion of mannose label to fucose occurs in wild-type and variant cell lines and that this pathway may be of greater importance in the variant cells; this result could explain at least in part, the higher than expected levels of ¹⁴C-label in glycoprotein in the variant cell lines. The changes in the glycosyl transferase activities in these lectin-resistant cell lines are probably involved in determining the concanavalin A-resistant property and the accompanying complex phenotype exhibited by these variant cell lines.     

Antigen-initiated B lymphocyte differentiation. IX. Characterization of memory AFC progenitors by buoyant density and sedimentation velocity separation.

The characteristics of memory B cell antibody-forming cell (AFC) progenitors from long-term hapten-primed CBA mice were investigated by using sedimentation velocity and buoyant density separation to isolate physically distinct B cell sub-sets. The isolated fractions were assayed by the adoptive immune response to NIP-POL antigen, under conditions where neither T cells nor other accessory cells were limiting the IgM or IgG AFC responses. The results were compared to previous studies on the IgM AFC-progenitors of unprimed adult mice. Splenic IgM and IgG memory AFC-progenitor activity was largely found among the typical B cells of slow to medium sedimentation rate, in contrast to the fastre sedimenting IgM AFC-progenitor activity of unprimed animals. Splenic IgM and IgG memory AFC-progenitor activity was found among the medium to light density cells, and so resembled by this parameter the IgM AFC-progenitor activity in unprimed animals. Thoracic duct lymphocytes from hapten-primed mice also exhibited memory IgM and IgG AFC-progenitor activity in the slow-medium sedimentation range. However, in contrast to spleen, the IgM and IgG memory AFC-progenitor activity in lymph was found among very dense B cells. Two physically distinct sub-populations of memory B cells have thus been identified, namely: i) small, medium-light density, presumably tissue-resident B lymphocytes found in spleen; and ii) small, dense, presumably recirculating B lymphocytes found in lymph. Both physical forms include IgM and IgG progenitors. Both forms are distinct from the larger, medium-light density "virgin" AFC-progenitors in the spleen of unprimed adult mice.     

Membrane fatty acid changes during the cell cycle of CV-1 cells

Monolayers of CV-1 cells were synchronized at the G1/S boundary of the cell cycle by a 24-h 2 mM thymidine blockade. Uptake of tritiated thymidine indicated that the peak DNA synthesis occurred 6-8 h after release from the block and that cell cycle time was 18-20 h. The fatty acid composition of phospholipids extracted from cells at 0, 7, and 18 h postblockade was measured by gas chromatography. The results indicate cyclic changes in membrane fatty acids with a significant increase in long-chain polyunsaturated fatty acids during the DNA synthesis phase (S phase) of the cell cycle.     

An improved function for fitting sedimentation velocity data for low-molecular-weight solutes.

Many traditional approaches to the analysis of sedimentation velocity data work poorly with data for low-molecular-weight solutes, which have sedimentation boundaries that are severely broadened by diffusion. An approach that has previously had some success is to directly fit these broad boundaries to approximate solutions of the Lamm equation that directly account for the high diffusion. However, none of the available approximate solutions work well at times both early and late in the run, or give boundary shapes that are highly accurate, especially for species of molecular weight < 10,000. An improved fitting function has been developed to overcome some of these limitations. The new function adds two correction terms to the Fujita-MacCosham solution. The optimum coefficients for these new correction terms were determined by a least-squares approach. The accuracy and limitations of fitting with this new function were tested against synthetic data sets obtained by finite-element methods, for analysis of samples containing either single species or several noninteracting species. We also compare the strengths and weaknesses of this method of analysis, and its ability to work with noisy data, relative to recently developed time-derivative methodologies.     

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.     

B lymphocyte subpopulations separated by velocity sedimentation. II. Characterization of tolerance susceptibility.

Neonatal and adult splenic B lymphocyte subpopulations, separated by velocity sedimentation, were tested in an in vitro splenic focus assay for their susceptibility to tolerance induction with hapten-protein conjugates. At least two tolerizable B cell subsets have been defined in the neonatal spleen; one comprising the slowly sedimenting, small lymphocytes and the other comprising the very rapidly sedimenting, large lymphocytes. In addition, a rapidly sedimenting large B cell subset in the adult spleen was found to be highly susceptible to tolerance induction. It was suggested that the inability to detect this susceptibility in unfractionated adult spleen was due to the low proportion of these very large lymphocytes in the total spleen cell population. The tolerizable B cell subsets presently defined probably represent the least mature B lymphocytes detectable in the splenic focus assay.     

An outline of functional self-organization in V1: synchrony, STLR and Hebb rules

A model of self-organization of synapses in the striate cortex is described, and its functional implications discussed. Principal assumptions are: (a) covariance of cell firing declines with distance in cortex, (b) covariance of stimulus characteristics declines with distance in the visual field, and (c) metabolic rates are approximately uniform in all small axonal segments. Under these constraints, Hebbian learning implies a maximally stable synaptic configuration corresponding to anatomically and physiologically realistic ‘‘local maps’’, each of macro-columnar size, and each organized as Möbius projections of a “global map” of retinotopic form. Convergence to the maximally stable configuration is facilitated by the spatio-temporal learning rule. A tiling of V1, constructed of approximately mirror-image reflections of each local map by its neighbors, is formed. The model supplements standard concepts of feed-forward visual processing by introducing a new basis for contextual modulation and neural network identifications of visual signals, as perturbation of the synaptic configuration by rapid stimulus transients. On a long time-scale, synaptic development could overwrite the Möbius configuration, while LTP and LTD could mediate synaptic gain on intermediate time-scales.     

An improved method for the separation of cells by sedimentation at unit gravity.

An apparatus is described for the separation of cells by sedimentation velocity at lg. The viscosity of the sample was raised by the addition of polyethylene oxide and subsequently the sample was layered on top of a density gradient via a sieve. With the aid of this procedure the various ploidy classes of rat-liver cells were enriched and murine leukemia cells were separated according to the phases of their life cycle.     

Sedimentation velocity separation: a preparation method for cervical samples.

A preparation procedure, aiming at monolayer deposition of cervical exfoliative material on glass slides for high resolution prescreening has been developed. The main features of this procedure are centrifugal deposition after suspension and sedimentation of samples over isopycnic medium of 1.026 density. Fractioning of the separation column after centrifugation at 50 X g yields two preparations with leukocytes, bacteria and cellular debris predominantly located on the first slide and epithelial cells on the second one. The degree of spatial cellular isolation as well as the amount of diagnostically relevant cells per slide seem to fit the requirements of automated high resolution analysis.     

An improved approximate solution of the Lamm equation for the simultaneous estimation of sedimentation and diffusion coefficients from sedimentation velocity experiments

Sedimentation and diffusion coefficients are important parameters to describe size and shape of macromolecules in solution. The data can be obtained from sedimentation velocity experiments by a nonlinear fitting procedure using approximate solutions for the Lamm equation. Here, we present a modification of such a model function that was originally proposed by Fujita [H. Fujita, Mathematical Theory of Sedimentation Analysis, Wiley, New York, 1962]. The extended model function is well suitable to study low molecular mass compounds. The improvement of this solution given here is based on using an adjustable value for the explicit integration variable, z, the reduced radius. This modification leads to more accurate sedimentation and diffusion coefficients compared to using a constant value of 0.5 as used by Fujita. The advantage of our modification was demonstrated by the analysis of noise-free curves calculated using the finite element method, as well as experimental curves obtained for the peptides angiotensin I and II. The relatively low sedimentation and diffusion coefficients found for both substances indicate that the peptides exist as extended chains of about 3.65 nm (angiotensin I) or 3.04 nm length (angiotensin II) in solution. The lack of higher-order structure of the peptides that was derived also from CD spectra might facilitate receptor binding, and could be one reason for the fast proteolytic digestion of the free peptides.     

Isolation and separation of highly enriched fractions of viable mouse gastric parietal cells by velocity sedimentation

Methods of tissue dissociation and cell separation have been modified to obtain highly enriched fractions of mouse gastric parietal cells. Suspension of gastric mucosal cells are prepared by pronase digestion of the glandular portion of the stomach from adult mice. By utilizing the velocity sedimentation technique to separate cells of different sizes it is possible to recovery parietal cells, which are larger than the other cell types, in fractions with purity of 75-95%. The homogeneity of cell fractions has been assessed by light and electron microscopy. The ability of the isolated cells to exclude the dye trypan blue, to incorporate labeled substrate, to consume oxygen, and to retain their structural integrity indicates that they are viable and still capable of functional activity.     

Screening of five specific cell cycle inhibitors using a T Cell lymphoma cell line synchrony/release assay

To obtain different cell populations at specific cell cycle stages, we used a cell culture synchronization protocol. Effects of five different cell cycle inhibitors acting throughout the cell cycle were examined by DNA flow cytometric analysis of a synchrony/release lymphoma cell line (CEM). The screening synchronized protocol showed that staurosporine, mimosine and aphidicolin are reversible G1 phase inhibitors that act at different times. Staurosporine acted in early G1, exhibited the strongest cytotoxic effect, and induced apoptosis. Mimosine and aphidicolin acted in late G1 and at the G1/S boundary, respectively. Hydroxyurea arrested CEM cells in early S phase, but later than the aphidicolin arrest point. Nocodazole synchronized CEM cells in M phase. All the inhibitors examined in this study can be used to synchronize cells at different phases of the cell cycle and were reversible with little toxicity except for staurosporine which is highly toxic. Because the regulatory mechanism of the cell cycle is disrupted by their effects on protein synthesis, however, these drugs must be used with caution.     

Identification and interpretation of complexity in sedimentation velocity boundaries.

Synthetic sedimentation velocity boundaries were generated using finite-element solutions to the original and modified forms of the Lamm equation. Situations modeled included ideal single- and multicomponent samples, concentration-dependent samples, noninteracting multicomponent samples, and reversibly self-associating samples. Synthetic boundaries subsequently were analyzed using the method of van Holde and Weischet, and results were compared against known input parameters. Results indicate that this analytical method provides rigorous diagnostics for virtually every type of sample complexity encountered experimentally. Accordingly, both the power and utility of sedimentation velocity experiments have been significantly expanded.     

Subcategory assessment method for social life cycle assessment. Part 1: methodological framework

Purpose

The aim of this work is to propose an objective method for evaluating subcategories in social life cycle impact assessment (S-LCIA). Methods for assessing subcategories have been available since 2006, but a number of these either fail to include all the subcategories envisaged in the guidelines for S-LCA (UNEP/SETAC 2009) or are subjective in their assessment of each subcategory.

Methods

The methodology is characterized by four steps: (i) the use of the organization as unit process, in which it was decided to assess the social profile of the organization responsible for the processes involved in the product life cycle, (ii) definition of the basic requirement to assess each subcategory, (iii) definition of levels based on the environment context or organizational practice and the data availability and (iv) assignment of a quantitative value.

Results and discussion

The result of the method applied was the development of the subcategory assessment method (SAM). SAM is a characterization model that evaluates subcategories during the impact assessment phase. This method is based on the behaviour of organizations responsible for the processes along the product life cycle, thereby enabling a social performance evaluation. The method, thus, presents levels for each subcategory assessment. Level A indicates that the organization exhibits proactive behaviour by promoting basic requirement (BR) practices along the value chain. Level B means that the organization fulfils the BR. Levels C and D are assigned to organizations that do not meet the BR and are differentiated by their context. The greatest difficulty when developing SAM was the definition of the BR to be used in the evaluation of the subcategories, though many indications were present in the methodological sheets.

Conclusions

SAM makes it possible to go from inventory to subcategory assessment. The method supports evaluation across life cycle products, thereby ensuring a more objective evaluation of the social behaviour of organizations and applicable in different countries.

Recommendations

When using SAM, it is advisable to update the data for the context environment. The method might be improved by using data for the social context that would consider not only the country, but also the region, sector and product concerned. A further improvement could be a subdivision of the levels to better encompass differences between organizations. It is advisable to test SAM by applying it to a case study.