Manometric determination of biological degradability of substances poorly soluble in aqueous environments

Several ways or methods of testing the biological degradability of organic substances poorly soluble in aqueous environments were verified by applying manometric BOD measurements. Model substrates employed here were dibutyl phthalate, dioctyl adipate, bio-fuel and some of their components. Dosages of substrates were varied by combining direct weighing and sonification, by using an emulsion with Triton X100, or by a solution in 1,1,2-trichlorotrifluoroethane. These selected variants of dosage, except for the last, proved to be equivalent from the viewpoint of degree and rate of biological degradation. The applied methods gave values of biochemical/theoretical oxygen demand ratio (BOD/ThOD) around 50%, even for substrates claimed to be biologically easily degradable.     

Cell Swelling Activates Phospholipase A2 in Ehrlich Ascites Tumor Cells

Ehrlich ascites tumor cells, loaded with ³H-labeled arachidonic acid and ¹⁴C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of ³H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of ¹⁴C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A₂ is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in ¹⁴C-labeled stearic acid release nor in the synthesis of phosphatidyl ¹⁴C-butanol in the presence of ¹⁴C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of ¹⁴C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A₁, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of ³H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP_βS or with AACOCF₃, an inhibitor of the 85 kDa, cytosolic phospholipase A₂. Based on these results we propose that cell swelling activates a phospholipase A₂—perhaps the cytosolic 85 kDa type—by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response. Received: 23 July 1996/Revised: 17 June 1997     

Immunodominant CD4+ T-cell epitope within nonstructural protein 3 in acute hepatitis C virus infection.

In acute hepatitis C virus infection, 50 to 70% of patients develop chronic disease. Considering the low rate of spontaneous viral clearance during chronic hepatitis C infection, the first few months of interaction between the patient's immune system and the viral population seem to be crucial in determining the outcome of infection. We previously reported the association between a strong and sustained CD4+ T-cell response to nonstructural protein 3 (NS3) of the hepatitis C virus and a self-limited course of acute hepatitis C infection. In this study, we identify an immunodominant CD4+ T-cell epitope (amino acids 1248 to 1261) that was recognized by the majority (14 of 23) of NS3-specific CD4+ T-cell clones from four of five patients with acute hepatitis C infection. This epitope can be presented to CD4+ T cells by HLA-DR4, -DR11, -DR12, -DR13, and -DR16. HLA-binding studies revealed a high binding affinity for 10 of 13 common HLA-DR alleles. Two additional CD4+ T-cell epitopes, amino acids 1388 to 1407 and amino acids 1450 to 1469, showed a very narrow pattern of binding to individual HLA-DR alleles. Our data suggest that the NS3-specific CD4+ T-cell response in acute hepatitis C infection is dominated by a single, promiscuous peptide epitope which could become a promising candidate for the development of a CD4+ T-cell vaccine.     

Dynamics of the cytoskeleton inAmoeba proteus: II. Influence of different agents on the spatial organization of microinjected fluorescein-labeled actin

Summary Iodoacetamido-fluorescein-(IAF)-labeled actin was microinjected into normal locomotingAmoeba proteus. Thereafter (30–60 minutes) changes in the cytoplasmic fluorescence distribution pattern and contractile activity were induced by internal and external chemical stimulation. Different agents such as phalloidin, procaine, 2.4-dinitrophenol (DNP), puromycin, ouabain and n-ethyl maleimide (NEM) interfere with the excitation-contraction mechanism involved in ordered pseudopodium formation during ameboid movement and cause various morphogenetic reactions based on actin polymerization-depolymerization cycles. Most frequent changes are (a) local condensation of IAF-actin and formation of a continuous IAF-actin layer at the cytoplasmic surface of the cell membrane and around the pulsating vacuole, (b) immobilization and hyalo-granuloplasm separation by combined contraction and detachment of the IAF-actin layer from the cell membrane, (c) organized and disorganized formation of pseudopodia by local contraction and disintegration of the IAF-actin layer, and (d) alterations in the rheological properties of the protoplasmic matrix by changes in the molecular state of soluble actin not incorporated into the cytoskeleton. The experimental approaches to the function of the actomyosin system in large amebas attainable by the method ofin vivo molecular cytochemistry are discussed in detail with respect to the participation of the cytoskeleton in motive force generation for cytoplasmic streaming and ameboid movement.     

Insulin receptors in 3T3 fibroblasts : Relationship to growth phase, transformation and differentiation into new cell types

The amount of [¹²⁵I]insulin binding per 2 × 10⁶ cells is measured in three lines of mouse embryonic 3T3 fibroblast at different growth stages. Insulin binding is found to be lowest in growing cells of all three types, increasing as cells reach stationary phase. Binding in 3T3-M cells approaches a plateau as cells become stationary. Insulin binding in 3T3-L cells, many of which differentiate into adipocytes following cessation of growth, show further increase in insulin binding post-confluence, in parallel with their differentiation into adipocytes. Binding of insulin in spontaneously transformed cells is higher at all phases of growth than the other two lines, rising to a much higher eventual plateau at approx. 17 days post-confluence. Scatchard plots of insulin binding tend to reflect the same degree of relative insulin binding in these three cell lines. Previously starved cells of all three types exhibit a drop in insulin binding following their first feeding, which corresponds with a second growth spurt in response to nutrients in fresh serum. These results suggest that insulin, as reflected by binding per cell, may play only a minor role in actively growing adequately fed cells of all three types, its major role developing as these cells approach confluence. It is also suggested that higher insulin binding in transformed vs non-transformed cells may indicate a special role for insulin in the loss of contact inhibition, by preserving transport of limiting nutrients in dense, nutrient-depleted transformed cultures.     

Suppressor T cells arising in mice undergoing a graft-vs-host response.

We investigated the ability of mice to generate antibody-forming cells when undergoing a graft-vs-host reaction. (C57BL/6 X DBA/2)F1 mice (BDF1) injected with C57BL/6 spleen cells generated suppressor T cells which inhibit antibody synthesis by BDF1 spleen cells in vitro. These T cells arose from the donor inoculum. They differ from helper T cells in size and they act directly on antigen reactive B cells. The suppressor T cells were specifically directed against components of the H-2 region of the reciprocal parental strain (DBA/2 = H-2d) in the hybrid F1 mouse.     

Bacteriorhodopsin,boundary lipid and protein conformers. A spin label study

A spin label study, as a function of temperature, has been made with the bacteriorhodopsin membrane using a stearic acid spin label. The ESR spectra show a strong variation with temperature and the presence of isosbestic points. The spectra are interpreted as indicating the presence of a two-component system with an activation energy (approx. 14 kcal/mol) corresponding to a protein conformational change. This activation energy is similar to that deduced from recent flash photolysis studies.It is concluded that the spin label is sensitive to the temperature-dependent protein conformational change in this membrane system.