Hydrogen peroxide-mediated protein oxidation in young and old human MRC-5 fibroblasts

It is suggested that the aging process is dependent on the action of free radicals. One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The present investigation was undertaken to reveal the proliferation-related changes in the protein oxidation and proteasome activity during and after an acute oxidative stress. It could be demonstrated that the activity of the cytosolic proteasomal system declines during proliferative senescence of human MRC-5 fibroblasts and is not able to remove oxidized proteins in old cells efficiently. Whereas in young cells removal of oxidized proteins was accompanied by an increase in the overall protein turnover, this increase in protein turnover could not be seen in old MRC-5 fibroblasts. Therefore, our studies demonstrate that old fibroblasts are much more vulnerable to the accumulation of oxidized proteins after oxidative stress and are not able to remove these oxidized proteins as efficiently as young fibroblasts.     

Flow-cytometric analysis of cytokine production in human paracoccidioidomycosis

Human infection with Paracoccidioides brasiliensis may result in three major outcomes: paracoccidioidomycosis-infection (PI), which is observed in healthy carriers living in endemic areas and the adult form (AF) and juvenile form (JF) of the disease. In this study we proposed to examine the intracellular expression of IFN-gamma, TNF-alpha, IL-2, IL-10, IL-12, CXCL8, CXCL9 and CXCL10 by peripheral blood mononuclear cells (PBMC) of patients with the JF and AF of the disease, as well as of PI individuals stimulated with PMA plus ionomycin, LPS or anti-CD3 plus anti-CD28, by flow cytometry. The results showed that PI individuals present a higher percentage of cells producing IFN-gamma, TNF-alpha, IL-2, CXCL9 and CXCL10 when compared to AF and JF patients. IFN-gamma was predominantly detected in CD3(+)CD8(+) T cells, whereas IL-2 and TNF-alpha were mainly expressed in CD3(+)CD4(+) cells. Monocytes of PI individuals also presented higher expression of CD80 and lower expression of CD86 when compared to JF and AF patients, and higher expression of HLA-DR, only when compared to JF patients. These results indicate that the differential production of cytokines and chemokines, as well as the expression of co-stimulatory molecules involved in antigen presentation, may influence the outcome of PCM infection.     

On the Y fluorescence in human male fibroblasts

Summary In a system of mitotically synchronized human fibroblast cells of the karyotype 46, XY and 47,XYY, the frequency and the variation in structure of the quinacrine dihydrochloride stained heterochromatic part of the Y chromosome was counted stepwise in regard to the periods of the cell cycle. There was no change of frequency and structure in correlation to these periods, especially no visible uncoiling in relation to the time of DNA synthesis.

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Zusammenfassung In einem System teilungssynchroner Kulturen menschlicher Fibroblasten des Karyotyp 46,XY und 47,XYY wurden Häufigkeiten und Strukturen des mit Quinacrindihydrochlorid gefärbten heterochromatischen Anteils des Y-Chromsoms in Abhängigkeit von den einzelnen Phasen des Zellcyclus fortlaufend beobachtet. Es fand sich keine Veränderung in Korrelation zu den Cyclusphasen, insbesondere keine meßbare Entspiralisation des heteropyknotischen F-body in Verbindung mit dem Zeitraum des DNS-Synthese.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

DNA-binding proteins in young and senescent normal human fibroblasts

DNA-binding proteins (DBP) from normal human diploid cells, strain WI38, were isolated by DNA-cellulose chromatography using undenatured calf thymus DNA. The DBP in the 0.15 M NaCl eluate were fractionated by polyacrylamide gel electrophoresis. Comparisons of the amounts of the DBP in different cell populations were made by labelling the cells with either ³H- or ¹⁴C-amino acid precursors for 40 h prior to pooling the cells for co-isolation of their DBP. When WI38 cells in the replicative and stationary phases were compared, five proteins, P5b (87 000 D), P6a (50 000 D), P8 (33 000 D), P9 (28 000 D) and P10 (25 000 D) were labelled to a greater extent in the replicating cells and two proteins, P5c (72 000 D) and P12 (18 000 D) were labelled to a greater extent in the stationary phase cells. In addition, several high molecular weight DBP, partially characterized as collagen and protocollagen, were preferentially labelled in the stationary phase cells. Stationary phase senescent WI38 cells at or near the end of their in vitro lifespan characteristically showed an increased proportion of protein component P8 (33 000 D) relative to stationary phase WI38 cells at early population doubling levels. Further characterization of WI38-P8 showed that it binds preferentially to single-stranded DNA and amounts to greater than 1% of the total soluble protein in young cells in growth phase. Thus WI38-P8 appears to be comparable to the P8 protein studied by Tsai & Green [27] in mouse 3T6 and human SB cells. The component which is increased in senescent or terminal phase non-dividing cell populations is judged to be the P8 protein by its position in SDS-gels and its preferential binding to single-stranded DNA.     

Flow-cytometric detection of changes in the fluorescence emission spectrum of a vital DNA-specific dye in human tumour cells

A multiparameter flow cytometric technique has been used to detect changes in the emission spectrum of the DNA-specific fluorochrome Hoechst 33342 during uptake by intact, human tumour cells and during the in vitro titration of permeabilized cells. The spectral shift phenomenon was associated with changes in dye: DNA ratio revealing heterogeneity in dye-binding sites. The degree of spectral shift was sensitive to changes in pH within the physiological range. Surprisingly, chromatin structure, in terms of DNase accessibility, was not a major factor in the generation of the spectral shift. The technique of fluorescence emission analysis permits cells with similar DNA contents to be distinguished on the basis of changes in the microenvironment of chromatin for both fresh and freezer-stored biopsy or experimental preparations.     

Subcellular fractionation of WI-38 fibroblasts: Compatison between young and old cells

WI-38 fibroblasts cultivated in vitro were homogenized and their subcellular organelles analysed by the techniques of differential centrifugation and isopycnic equilibration in density gradient. In these experiments, the assayed enzymes were known to be specifically associated with subcellular components in other cells types. In most cases, their behaviour and properties corresponded with observations made in earlier studies and we could consider them as being representative of the specific subcellular organelles.Some significant differences were observed between young and old fibroblasts. The specific activity of alkaline phosphodiesterase was lower in the old cells whereas for the other enzymes it was identical or higher, especially for the 5′-nucleotidase; also the particulate fractions obtained by differential centrifugation contained more material. After equilibration in density gradient, the average density of the 5′-nucleotidase, alkaline phosphodiesterase and $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-glucosaminidase}$ was less in the old than in the young cells, whereas that of the galactosyltransferase of Golgi apparatus was greater. For mitochondria, endolasmic reticulum and peroxisomes, the differences observed were small.     

Flow-cytometric analysis of mouse pritoneal macrophages

Flow-cytometric analysis of mouse peritoneal macrophages (MΦ) stained with acridine orange defined three populations with increasing RNA content. Resident MΦ displayed variable bimodal distributions of low and intermediate cellular RNA content with high RNA content only observed after in vitro stimulation with fetal calf serum or/and bacterial lipopolysaccharide (LPS). In contrast, only few resident macrophages from the LPS nonresponder strain C3H/HeJ increased in RNA content upon LPS stimulation. Macrophages with high RNA content developed transiently after in vivo stimulation with either thioglycollate broth or paraffin oil. Proteose peptone-elicited macrophages transformed only after additional in vitro stimulation with fetal calf serum and/or LPS. The magnitude of the in vitro response upon stimulation, assessed as increasing percentage of either intermediate or high RNA macrophages, was dependent on the composition of the MΦ population at the onset of in vitro culture. Effective increase in cellular RNA content was always paralleled by improved adherence of plated macrophages to the culture vessel. In conclusion, flow-cytometric measurement of macrophage populations might become a useful tool to quantify macrophage activation or stimulation.     

Heterogeneity of dimer excision in young and senescent human dermal fibroblasts

We have examined the relationship between nucleotide excision of the main UV-induced photoproduct, the cyclobutane pyrimidine dimer and in vitro cellular senescence. An in situ semiquantitative immunocytochemical assay has demonstrated that, following a UV-C dose of 15 J m-2, young human dermal fibroblasts maintained in a high level of serum are more efficient than senescent fibroblasts in the removal of dimers. However, in G0-arrested cultures (serum-starved), young fibroblasts are compromised in their ability to remove dimers and are significantly less efficient than senescent cells in this process. Supplementation of the culture medium with 0.1 mm deoxyribonucleosides enhances the removal of dimers in both young and senescent fibroblasts in proliferating or serum-starved cells. These data indicate that overall there is a modest but significant reduction in nucleotide excision of dimer photoproducts in cells as they age in vitro. In addition, G0-arrested young cells exhibit reduced removal of dimers, although this can be complemented by deoxyribonucleoside addition. In addition, this in situ assay has revealed heterogeneity in both susceptibility to UV-C-induced damage and excision. Overall, we provide evidence of reduced UV-induced damage excision in senescent compared with young fibroblasts, and demonstrate modulation of these processes in young and senescent cells under specific growth conditions.     

Voltage-dependent calcium channels in young and old human red cells

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of α_1C, subunit of high-voltage activated Ca channels (pan α₁) and against conserved residues of α_1C and α_1E subunits. No specific reaction was detected with antibodies against conserved residues of α_1A, α_1B, or α_1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan α_1A or anti-α_1E blots, whereas two bands (200 and 230 kDa) were detected by α_1C exposure, Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers' under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl+5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15–50% reductions of ⁴⁵Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Q-and N-type blockers also reduced Ca influx to various extents (25–60%). Old cells were generally insensitive to L-type but not to non-L-type, blockers. Raising external K to about 70–80 mM reduced by 50–100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results, clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.     

Vitamin E in young and old human red blood cells

Young and old human red blood cells contain about the same amount of alpha-tocopherol, a compound which has previously been shown to be the major lipid-soluble, chain-breaking antioxidant present in such cells. Since red blood cells lose up to ca. 20% of lipid material from their membrane as they age, the alpha-tocopherol/membrane-lipid ratio actually rises with age rather than declining as might have been expected on the basis of the free radical theory of aging. The alpha-tocopherol/arachidonic acid moiety ratios increase in the order: young red blood cells less than old red blood cells less than plasma, which argues against the suggested membrane stabilizing effect of alpha-tocopherol/arachidonic acid moiety complexes.     

Differences in intramembrane particle distribution in young and old human erythrocytes

The distribution of intramembrane particles in human erythrocytes was studied by freeze-fracture on young and old cells and compared to that obtained after ATP depletion or following addition of a clustering agent. It was shown that intramembrane particles became aggregated and the mean particle density increased as the cells aged. Likewise, both particle aggregation and increased density were found in young cells after moderate ATP depletion. In contrast, mean particle density was markedly reduced in both cell types after exhaustive depletion. Paradoxically, Zn treatment led to decreased particle density in young cells, whilst producing the opposite effect in aged cells. The results suggest that their low ATP content may account for the increased particle density of senescent cells.     

Acetylcholinesterase inactivation in young and old human red blood cells.

A comparison is made of the effects of certain enzyme-inactivating agents on the acetylcholinesterase (ACHE) of young and old human erythrocytes. Normal red cells and ACHE-deficient red cells are separated in accordance with their density and then exposed to trypsin, cephalothin and tannic acid. The ACHE activity of young and old cells is affected to the same extent, indicating that inactivation is independent of cullular age and the initial enzyme activity.     

The fluorescence and bright field microscopic demonstration of cathepsin B in human fibroblasts

Summary Cathepsin B was demonstrated cytochemically in human fibroblasts with Z-Ala-Arg-Arg-2-(4-methoxy)naphtylamide as substrate. The enzyme was visualized in the bright field microscope with the diazonium salt Fast Blue B as coupling reagent and in the fluorescence microscope with 5-nitrosalicylaldehyde. With both methods cathepsin B was found in small granules distributed throughout the cytoplasm.     

The fluorescence and bright field microscopic demonstration of cathepsin B in human fibroblasts

Cathepsin B was demonstrated cytochemically in human fibroblasts with Z-Ala-Arg-Arg-2-(4-methoxy)naphtylamide as substrate. The enzyme was visualized in the bright field microscope with the diazonium salt Fast Blue B as coupling reagent and in the fluorescence microscope with 5-nitrosalicylaldehyde. With both methods cathepsin B was found in small granules distributed throughout the cytoplasm.     

Morphological analysis of young and old pellicles of Salmonella Typhimurium

Abstract

A wide variety of microorganisms are able to form biofilms at the interface between air and liquid (pellicles). In this study changes during the maturation of the pellicle of Salmonella Typhimurium were analysed and the role of cellulose in the pellicle structure and morphology evaluated. The morphology of both sides of the pellicle was characterised using atomic force microscopy and scanning electron microscopy. Overall, there was a marked difference in the morphology of the water-facing (WF) and air-facing (AF) biofilm surfaces. While the AF side appeared to be uniform, and extensively covered with an exocellular coating, cells in the WF side were distributed into clusters and were less covered. However, the similarity in size and shape of single cells from both sides of the pellicle may indicate that the bacterial cells across the pellicle have a similar physiological status. During maturation, porous structures with multiple cracks and channels were created in the pellicle, leading to disintegration. By comparison with the structure of pellicles of a cellulose-deficient mutant, it was demonstrated that the observed disintegration of mature pellicles probably occurred in part by self-hydrolysis of components of the matrix.     

Morphological analysis of young and old pellicles of Salmonella Typhimurium

A wide variety of microorganisms are able to form biofilms at the interface between air and liquid (pellicles). In this study changes during the maturation of the pellicle of Salmonella Typhimurium were analysed and the role of cellulose in the pellicle structure and morphology evaluated. The morphology of both sides of the pellicle was characterised using atomic force microscopy and scanning electron microscopy. Overall, there was a marked difference in the morphology of the water-facing (WF) and air-facing (AF) biofilm surfaces. While the AF side appeared to be uniform, and extensively covered with an exocellular coating, cells in the WF side were distributed into clusters and were less covered. However, the similarity in size and shape of single cells from both sides of the pellicle may indicate that the bacterial cells across the pellicle have a similar physiological status. During maturation, porous structures with multiple cracks and channels were created in the pellicle, leading to disintegration. By comparison with the structure of pellicles of a cellulose-deficient mutant, it was demonstrated that the observed disintegration of mature pellicles probably occurred in part by self-hydrolysis of components of the matrix.     

Creatine and creatinine transport in old and young human red blood cells

The time course of creatine influx or efflux as measured in populations of red cells or red cell ghosts with normal age distribution does not follow simple two-compartment kinetics. This suggests that the contributions of individual cells to transport as measured in the populations as a whole are not uniform. In agreement with this inference, fractionation of red cell populations with respect to cell age shows that transport in young cells is considerably faster than in old cells.The dependence of creatine transport on creatine concentration in the medium follows an equation that can be interpreted to represent a super-imposition of a saturable component (apparent K_m = 0.02 mM) and another component that cannot be saturated up to a creatine concentration of 5.0 mM. In contrast to the non-saturable component, the saturable component depends on the energy metabolism of the cell and can be inhibited by β-guanidinopropionic acid and the proteolytic enzyme pronase. This latter finding suggests that the saturable component represents active transport that is mediated by a transport protein. The non-saturable component is little, if at all, dependent on cell age while the saturable component is higher in young cells than in old cells. Phloretin inhibits both components of creatine flux, but the maximal inhibition that can be achieved at high concentration is only 70–80%.Under the experimental conditions used for the study of creatine transport, creatinine equilibration between cells and medium follows the kinetics expected for a steady-state two-compartment system. Creatinine flux is proportional to creatine concentration over the concentration range studied (up to 5 mM). It cannot be inhibited by β-guanidinopropionic acid or pronase.     

Calcium pump phosphoenzyme from young and old human red cells

An increase in intracellular Ca(2+) occurs during ageing of human erythrocytes in vivo. The aged cells show a reduced capacity for active Ca(2+) extrusion. Such a defect may arise from pump proteolysis, due to calpain activation by the raised intracellular Ca(2+). To test this possibility, Ca(2+) pump phosphorylation by [gamma-(32)P]ATP was studied on percoll-separated young and old human erythrocytes. After phosphorylation for 30 s with Ca(2+), the amount of phosphoenzyme produced by the young cell membranes was 50% that of the old cells. With Ca(2+) plus La(3+), in contrast, the phosphoenzyme level was nearly the same in both preparations. After a prolonged phosphorylation period (50-90 s), the phosphoenzyme reached almost identical equilibrium levels in both membrane preparations. On the other hand, a single Ca(2+)-dependent radioactive band of about 150 kDa was apparent in both preparations after acidic electrophoresis. Likewise, Western blotting using 5F10 monoclonal antibody also detected a single band of similar molecular weight. These results demonstrate that there is no alteration in either molecular mass or number of active Ca(2+) pump units during cell ageing, thus indicating that the reduced Ca(2+) pumping activity of aged cells does not arise from pump proteolysis.