Role of cytoskeletal elements in the retractile activity of human skin fibroblasts

Giant axonal neuropathy skin fibroblasts, which are characterized by a selective and partial disorganization of vimentin filaments [1] exhibited, when compared with normal skin fibroblasts, less fibrin clot retractile (FCR) activity and spreading within the fibrin clot both during active growth and resting stage. Skin fibroblasts derived from patients affected with adenomatosis of the colon and rectum, which display a disorganized actin network [2], exhibited reduced FCR activity and spreading within the fibrin clot only during resting stage. FCR inhibition was also obtained by treating the cells with colcemid, cytochalasin B (CB) and dihydrocytochalasin B. The data suggest that FCR activity is under the control of different cytoskeletal structures. For the first time, a direct involvement of intermediate-sized filaments could be demonstrated in the interaction between fibroblasts and an organic substratum.     

Spatial propagation and localization of blood coagulation are regulated by intrinsic and protein C pathways, respectively

Blood coagulation in vivo is a spatially nonuniform, multistage process: coagulation factors from plasma bind to tissue factor (TF)-expressing cells, become activated, dissociate, and diffuse into plasma to form enzymatic complexes on the membranes of activated platelets. We studied spatial regulation of coagulation using two approaches: 1), an in vitro experimental model of clot formation in a thin layer of plasma activated by a monolayer of TF-expressing cells; and 2), a computer simulation model. Clotting in factor VIII- and factor XI-deficient plasmas was initiated normally, but further clot elongation was impaired in factor VIII- and, at later stages, in factor XI-deficient plasma. The data indicated that clot elongation was regulated by factor Xa formation by intrinsic tenase, whereas factor IXa was formed by extrinsic tenase on activating cells and diffused into plasma, thus sustaining clot growth. Far from the activating cells, additional factor IXa was produced by factor XIa. Exogenously added TF had no effect on the clot growth rate, suggesting that plasma TF does not contribute significantly to the clot propagation process in a reaction-diffusion system without flow. Addition of thrombomodulin at 3-100 nM caused dose-dependent termination of clot elongation with a final clot size of 2-0.2 mm. These results identify roles of specific coagulation pathways at different stages of spatial clot formation (initiation, elongation, and termination) and provide a possible basis for their therapeutic targeting.     

Flow-induced permeation of non-occlusive blood clots: an MRI study and modelling

The success of clot thrombolysis very much depends on efficient clot permeation with blood plasma carrying the thrombolytic agent. In this paper clot permeation was studied by dynamic magnetic resonance imaging (MRI) on artificial non-occlusive blood clots inserted in an artificial circulation system filled with blood plasma to which an MRI contrast agent was added. The MRI results revealed that clot permeation is much faster and more efficient at the entrance of the flow channel across the clot. Clot permeation with fluid was simulated numerically as well. The simulation was based on numerical solution of Navier-Stokes equations for the flow in the channel and within the clot. The clot was considered as a porous material with known permeability and porosity. Based on the calculated velocity profiles, concentration profiles of fluid in the clot were modelled. These agreed well with the MRI results. The presented model of clot permeation with fluid may also serve as a useful extension to numerical modelling of dissolution of non-occlusive blood clots during thrombolytic therapy.     

Evaluation of the reorganization in the high-order structure of DNA occurring during cell senescence

Electron microscopic pictures of loosened chromatin obtained from human fibroblasts aged in vitro were analyzed with an image processor. It was found that the density of the 30-nm chromatin fibers decreased progressively through the cell population lifespan. The spacing between the fibers increased abruptly during the last divisions (phase IV). The evolution of these 2 parameters, density and spacing, was identical, respectively, with that of the decline in the rate of initiating DNA synthesis after seeding the cells and with that of the maximal percent of cells synthesizing DNA during a 24-h period between seeding and resting phase. Cells from 2 human donors (30 and 60 years old) with Werner's syndrome were compared, using the same methodology, with those of 9 normal donors ranging from 29 to 98 years old. The spacing between chromatin fibers in the Werner's cells was significantly increased as compared to the age-matched normal controls while the density was within the normal range. The results are discussed with regard to other modifications in DNA structure previously reported in the same cell system.     

Conversion of cobalt-free corrinoid to vitamin B12 with the resting cells of Rhodobacter sphaeroides

A cobalt-free corrinoid (CFC) isolated from Rhodobacter sphaeroides was converted to cobalamin intracellularly with the resting cells prepared from an aerobic culture on a cobalt-containing medium. The maximum conversion activity was observed with the cells harvested at early exponential growth phase. The CFC might be used as a peculiar precursor in cobalamin biosynthesis.     

Hemophilia A and B are associated with abnormal spatial dynamics of clot growth

To gain greater insight into the nature of the bleeding tendency in hemophilia, we compared the spatial dynamics of clotting in platelet-free plasma from healthy donors and from patients with severe hemophilia A or B (factor VIII:C or IX:C<1%). Clotting was initiated via the intrinsic or extrinsic pathway in a thin layer of nonstirred plasma by bringing it in contact with the glass or fibroblast monolayer surface. The results suggest that clot growth is a process consisting of two distinct phases, initiation and elongation. The clotting events on the activator surface and the preceding period free of visible signs of clotting are the initiation phase. In experiments with and without stirring alike, this phase is prolonged in hemophilic plasma activated by the intrinsic, but not the extrinsic pathway. Strikingly, both hemophilia A and B are associated with a significant deterioration in the elongation phase (clot thickening), irrespective of the activation pathway. The rate of clot growth in hemophilic plasma is significantly lower than normal and declines quickly. The resulting clots are thin, which may account for the bleeding disorder.     

Conversion of cellulose to sugars by resting cells of a mesophilic anaerobe, Bacteriodes cellulosolvens

During the growth of Bacteroides cellulosolvens in media containing cellulose, the accumulation of unutilized sugars in the culture broth occurred mainly during the stationary phase of growth. Cells harvested during the stationary phase of growth continued to convert both cellulose and hemicellulose to cellobiose, glucose, and xylose. These three sugars caused feedback inhibition. Continuous removal of these sugars during the incubation of cells with cellulose at pH 5 accumulated ca. 32 g/L of sugars as compared to ca. 17 g/ produced under batch conditions of growth. Sugar formation by resting cells also increased with increasing cell concentration and did not require any nutrient.     

Spatiotemporal dynamics of fibrin formation and spreading of active thrombin entering non-recalcified plasma by diffusion

The spatiotemporal dynamics of clot growth was studied in non-stirred non-recalcified plasma where thrombin entered by diffusion. Under these conditions, the clot rapidly grew for 30-45 min and then stopped growing on reaching 0.4-0.5 mm in size. The dynamics of clot growth and its size almost did not depend on the thrombin concentration in the range from 50 to 400 nM. FITC-thrombin was shown to permeate the growing clot. The clot size in antithrombin-deficient plasma increases with decreasing antithrombin concentration, being 1.5 mm in the plasma depleted of antithrombin to 5% of its initial level. The data on the spatial distribution of amidolytic activity in the growth zone of the clot suggested that thrombin was not the sole source of this activity. Analysis showed that this additional activity arising during thrombin diffusion into plasma was largely accounted for by thrombin-alpha(2)-macroglobulin complex.     

Acute infection with Epstein-Barr virus targets and overwhelms the peripheral memory B-cell compartment with resting, latently infected cells

In this paper we demonstrate that during acute infection with Epstein-Barr virus (EBV), the peripheral blood fills up with latently infected, resting memory B cells to the point where up to 50% of all the memory cells may carry EBV. Despite this massive invasion of the memory compartment, the virus remains tightly restricted to memory cells, such that, in one donor, fewer than 1 in 10(4) infected cells were found in the naive compartment. We conclude that, even during acute infection, EBV persistence is tightly regulated. This result confirms the prediction that during the early phase of infection, before cellular immunity is effective, there is nothing to prevent amplification of the viral cycle of infection, differentiation, and reactivation, causing the peripheral memory compartment to fill up with latently infected cells. Subsequently, there is a rapid decline in infected cells for the first few weeks that approximates the decay in the cytotoxic-T-cell responses to viral replicative antigens. This phase is followed by a slower decline that, even by 1 year, had not reached a steady state. Therefore, EBV may approach but never reach a stable equilibrium.     

Red cell and total blood volumes during sexual excitement and copulation in the boar.

Measurements of hematocrit, total plasma protein and red cell volume were made during sexual excitement and copulation in boars. Red cell volume (RCV) was determined by isotope dilution technique using endogenous 51Cr-tagged red cells. Basing on these data changes in the total blood volume (TBV) and plasma volume (PV) were calculated by 2 indirect methods. RCV increased by 12% to 16% in the intial phase of ejaculation and remained increased during ejaculation and 40 minutes after copulation. TBV and PV decreased during copulation, greatest drop being found in the final phase of ejaculation. After ejaculation the TBV was increasing, first to the resting value before copulation (about 20 minutes after copulation), thereafter it became markedly higher than during the resting period. Depending on the method used for calculation significant differences were found in the quantity of TBV and PV drop during ejaculation.     

The role of respiration in tumor cell transition from the noncycling to the cycling state

Resting Yoshida AH130 hepatoma cells, harvested at the plateau of tumor development in vivo, were recruited into the cycling state following transfer to an in vitro system whereby these cells were incubated in the autologous ascites plasma diluted with buffered saline and enriched with glucose. In this system, cell recruitment into the phase of DNA synthesis (S phase) strictly depends on the activity of the respiratory chain and is abolished by anaerobiosis as well as by antimycin A, although the intracellular levels of ATP and the rate of protein synthesis are practically unaffected by these treatments. Furthermore, 2,4-dinitrophenol, at concentrations which uncouple the respiratory phosphorylation and hence enhance both glycolysis and oxygen consumption, does not hinder cell promotion into S phase. Thus, the absolute respiration dependence of cycling resumption by resting ascites cells does not seem to rely on respiratory ATP supply, but rather is linked to the electron flow through the respiratory chain.     

PROTEIN METABOLISM IN TUMOR CELLS AT VARIOUS STAGES OF GROWTH IN VIVO

Protein metabolism of Yoshida ascites hepatoma cells was studied in the early phase of logarithmic proliferation and in the following stage in which cell mass remains constant (resting phase). The rate of protein synthesis was measured by a short-time incorporation of [⁸H]lysine, while degradation was concurrently assessed by following the decrease of specific activity of [¹⁴C]lysine-labeled proteins. Most of the labeled amino acid injected intraperitoneally into the animal was immediately available for the tumor cells, with only a minor loss towards the extra-ascitic compartment. It was thus possible to calculate the dilution of the isotope in the ascitic pool of the lysine, which increased concurrently with the ascitic plasma volume. Amino acid transport capacity did not change in the log vs. the resting cells. This fact permitted the correction of the specific activity of the proteins synthesized by tumors in the two phases, taking into account the dilution effect. Protein synthesis was found to proceed at a constant rate throughout each of the two phases, although it was 30% lower during the resting as compared to the log phase. When cell mass attained the steady-state, protein degradation occurred at such a level as to balance the synthesis. Throughout the resting phase the amount of lysine taken up by the cells and renewed from the blood remained unchanged. Protein turnover, as studied in subcellular fractions, exhibited a similar rate in nuclei and microsomes, where it proceeded at a higher level than in mitochondria. On the whole, the results encourage the use of the Yoshida ascites hepatoma as a suitable model for studying protein turnover in relation to cell growth in vivo.     

Changes in the Ultraviolet Sensitivity of Escherichia coli During Growth in Batch Cultures

The ultraviolet (UV) sensitivity of Escherichia coli B/r harvested at various times during growth in batch cultures was measured. The results showed a period of increased UV sensitivity in late log phase, just before the cultures entered stationary phase. This increase in sensitivity was associated with a decreased shoulder in the UV survival curves. The postirradiation division delay of survivors was shortest for cells harvested during the period of maximal sensitivity. This period of increased UV sensitivity during late log phase was not found in the radiation-sensitive, repair-deficient mutant B(s-1) (a strain which is unable to excise pyrimidine dimers from UV-damaged deoxyribonucleic acid). These results suggest that the variation in UV sensitivity of E. coli B/r as a function of time of harvesting of the cells from batch cultures is related to the varying capacities of these populations to repair UV-damaged deoxyribonucleic acid. Further experiments designed to elucidate the mechanism underlying this variation in UV sensitivity indicated that it arises from the partial depletion of nutrients in the medium during late log phase. We suggest that growth in such depleted media leads to a depression in the intercellular concentration or activity of one or more of the repair enzymes concerned with the repair of damaged deoxyribonucleic acid.     

Ca2+ removal mechanisms in rat cerebral resistance size arteries.

Tissue blood flow and blood pressure are each regulated by the contractile behavior of resistance artery smooth muscle. Vascular diseases such as hypertension have also been attributed to changes in vascular smooth muscle function as a consequence of altered Ca2+ removal. In the present study of Ca2+ removal mechanisms, in dissociated single cells from resistance arteries using fura-2 microfluorimetry and voltage clamp, Ca2+ uptake by the sarcoplasmic reticulum and extrusion by the Ca2+ pump in the cell membrane were demonstrably important in regulating Ca2+. In contrast, the Na+-Ca2+ exchanger played no detectable role in clearing Ca2+. Thus a voltage pulse to 0 mV, from a holding potential of -70 mV, triggered a Ca2+ influx and increased intracellular Ca2+ concentration ([Ca2+]i). On repolarization, [Ca2+]i returned to the resting level. The decline in [Ca2+]i consisted of three phases. Ca2+ removal was fast immediately after repolarization (first phase), then plateaued (second phase), and finally accelerated just before [Ca2+]i returned to resting levels (third phase). Thapsigargin or ryanodine, which each inhibit Ca2+ uptake into stores, did not affect the first but significantly inhibited the third phase. On the other hand, Na+ replacement with choline+ did not affect either the phasic features of Ca2+ removal or the absolute rate of its decline. Ca2+ removal was voltage-independent; holding the membrane potential at 120 mV, rather than at -70 mV, after the voltage pulse to 0 mV, did not attenuate Ca2+ removal rate. These results suggest that Ca2+ pumps in the sarcoplasmic reticulum and the plasma membrane, but not the Na+-Ca2+ exchanger, are important in Ca2+ removal in cerebral resistance artery cells.     

Morphometry of fetal Leydig cells in the monkey (Macaca fascicularis), correlation with plasma testosterone

The evolution of Leydig cells in Macaca fascicularis fetuses was followed throughout gestation (50-150 d) by morphometric procedures (volume densities of: cells, SER, mitochondria and lipid droplets). Testosterone from umbilical artery plasma was radioimmunoassayed starting on day 57. After predifferentiation and differentiation phases, Leydig cells entered the maturity phase (57-66 d), they occupied 19% of testicular volume, SER and lipid droplets represented 19% and 5% respectively of cytoplasmic volume. Then Leydig cells regressed dramatically (involution phase I: 66-83 d), their volume density decreased to 8%, that of SER to 12% whereas lipids doubled. Leydig cell volume density diminished to 5% during the second half of gestation (involution phase II), but their ultrastructure was not significantly altered. High plasma testosterone level (2.4 ng/ml) was observed during the maturity phase of Leydig cells, decline of testosterone occurred during involution phases I and II (1.13 and 0.58 ng/ml respectively). Its was shown that from day 57 to the end of fetal development the evolution of the plasma testosterone level correlated with the Leydig cell volume density and the SER volume density.     

The effects of starving versus fasting on blood composition in larval house crickets

Larval crickets (Acheta domesticus) starved for 2 days during the growth phase of the instar consumed twice as much water as larvae that ceased feeding of their own accord during the last 2 days of the last instar. The behaviour of drinking more water during starvation may compensate for dry weight loss and prevent the larvae from missing the critical weight required to initiate the next moult. During starvation the plasma volume increased while the tissue volume remained constant, which produced a shift in both organic and inorganic solutes from the tissues into the plasma. During fasting there was no change in tissue or plasma volume, therefore large osmotic adjustments were unnecessary, and the only change in plasma solutes noted was a decline in plasma proteins.The titres of proteins, lipids and amino acids remained constant during 2 days of starvation, though the amount of each increased because of the increased plasma volume. Although both the titre and the amount of plasma sugar sharply declined during starvation, there was no change in the sugar titre when the insects fasted. There was some evidence that prior to fasting the programmed gradual decline in food intake matched the decline in metabolic rate, which permitted a plasma sugar stability not evident in starved larvae. The decline in plasma proteins during the fasting phase appeared due to the removal of a larval specific protein and not a direct result of fasting.     

Evidence for the existence of "survival factors" as an explanation for some peculiarities of yeast growth, especially in grape must of high sugar concentration

The retardation and arrest of fermentation, observed before the complete sugar consumption of high-sugar grape must, come from an inhibition of the yeast metabolism during its decline phase and are variable with the strain. The addition of nutritional growth factors stimulates the initial growth of the yeast but is ineffective in the decline phase. Some substances, known previously as yeast anaerobic growth factors (sterols, oleanolic acid, oxytocin), in some conditions (initially aerated grape must and aerobically cultivated yeast) act by increasing the viability of the resting cells and prolonging their fermentation activity. These substances have been named "survival factors."     

Changes in protein content per cell during growth of mouse L cells

The relationship between cell density and protein content per cell was examined in monolayer and suspension cultures of mouse L cells. In monolayer cultures, the protein content per cell reached a maximum at 6 h after plating and retained this level for 18 h. Thereafter, the protein content per cell declined gradually during the exponential growth phase and finally returned to the initial level at the stationary phase. The changes were neither due to the effect of trypsinization nor to the exhaustion of the medium. The protein content per cell in a sparse culture was always greater than that in a dense culture for monolayer culture of L cells. In suspension culture the increase of protein content per cell during the lag phase was similar to that found in monolayer cultures. However, the gradual decline of protein content per cell observed during the exponential phase of monolayer cultures was not detected during that of a suspension culture. The results suggest that the decrease of protein content per cell in monolayer cultures may be related to some function of cell plasma membrane which could be inhibited by a cell-to-cell contact.     

Fibrin clot retraction by human skin fibroblasts: effects of ADP and thrombin.

Thrombin stimulated human skin fibroblasts to retract fibrin clots. When Bothrops marajoensis thrombinlike enzyme was substituted for thrombin, no retraction occurred. Fibroblasts were found to contain 12 nmole of ATP and 3.6 nmole of ADP/mg of protein, a value closely resembling that of nonmetabolic adenine nucleotides in platelets. Thrombin caused neither release of adenine nucleotides from the suspension of fibroblasts harvested enzymatically nor did addition of ADP stimulate fibroblasts to retract fibrin clots.