Swelling of hepatocytes injured by oxidative stress suggests pathological changes related to macromolecular crowding.

A fundamental property of living matter is the ability to establish and maintain order. Mild changes in cell volume have a role in metabolic control. Furthermore, cellular swelling is a way of living cells to react to a variety of stressors. Data from experimental pathology, biochemistry and biophysics and theoretical arguments from biology, biochemical evolution, cytology and biophysics are considered to attempt an integration of several current concepts on different subjects (intracellular compartmentation, cellular swelling, macromolecular crowding, perturbing and non-perturbing solutes). The purpose is to provide a framework for conceptualizing in modern terms the question whether cellular swelling induced by oxidative stress should be considered merely a cell adaptation balanced by antioxidant defenses and by other biochemical devices apt to preserve the intracellular environment and normal cell functioning, or whether swelling of high amplitude should be regarded as a true pathological change. The basic question dated 1982: "how crowded is the cytoplasm?" is a matter for discussion as far as swollen cells are concerned. This paper examines the liver for cellular swelling of high amplitude (about+30%) caused by iron or by thyroid hormone+iron (histological picture of "cloudy swelling") or the steatogenic poison CCl(4), also known as a source of oxyradicals, which causes an even more pronounced cellular swelling. In CCl(4)-toxic fatty liver the strong increase of tissue water is substantially masked by the parallel increase of tissue dry solids due to fat accumulation. This example of a "tissue dilution artefact" is discussed in connection with the increase of tissue water also in toxic fatty liver induced by white phosphorus and ethanol. In CCl(4)-toxic fatty liver the normal K(+)/Na(+) ratio (about 3) is substantially maintained, whereas the concentrations of the two cations ("perturbing osmolytes") in tissue water are noticeably decreased, a finding which was not further studied at the time the observations were made because biochemistry was not yet advanced enough to allow an explanation. Today, a logic hypothesis is that an increase of non-perturbing solutes such as taurine and betaine, maintains the physiological intracellular osmotic pressure and that the harmful effects of CCl(4) are limited because of the protective effects of these molecules and of molecular chaperones against damage by oxyradicals. However, as a consequence of cellular swelling, intracellular changes in ionic strength and macromolecular crowding should occur thus affecting enzyme activities. Models and techniques apt to investigate this problem experimentally are suggested.

Phenotypic alterations in senescent large-vessel and microvascular endothelial cells.

Endothelial cell senescence likely plays a key role in age-associated vascular diseases. A close relationship between in vitro and in vivo senescence of endothelial cells has been established. Therefore, elucidating the structural and functional changes occurring during long-term cultures of endothelial cells would contribute to clarifying the pathogenesis of vascular disorders in the elderly. We investigated the effects of replicative senescence on the architecture of bovine aortic vs microvascular endothelial cells. A marked increase in cell area was observed in both cell types, whereas dramatic morphological alterations were detected in microvascular endothelial cells only. The latter also showed age-associated reorganization of the actin cytoskeleton. Finally, both aortic and microvascular endothelial cells lost their migratory response to basic fibroblast growth factor with age. Our results highlight dramatic structural and functional alterations in senescent endothelial cells. Such rearrangements might account for in vivo endothelial cell alterations involved in age-associated vascular dysfunction.

Increment of nonreceptor tyrosine kinase Arg RNA as evaluated by semiquantitative RT-PCR in granulocyte and macrophage-like differentiation of HL-60 cells.

The products of the human Arg gene and human, mouse, Drosophila, and nematode Abl genes characterize the Abelson family of nonreceptor tyrosine protein kinase. The Arg gene, expressed as a 12-kb transcript, codes a protein highly related to c-abl in the tyrosine kinase, SH2, and SH3 domains, and both proteins have a myristoylated isoform. The C-terminal domains of Arg and c-abl, poorly similar to each other, may account for their different functions. Arg is cytoplasmic, c-abl also has nuclear localization, and their products have different transforming activity. To gain insight about the role of Arg in myeloid differentiation we investigated Arg gene expression in HL-60 cells differentiated with all-trans retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate. With a semiquantitative reverse transcriptase-polymerase chain reaction assay it was evident that the Arg transcript level in HL-60 cells differentiated toward granulocyte and macrophage-like lineage was, respectively, 3.5- and 2.8-fold the Arg level evidenced in undifferentiated HL-60 cells. In the HL-60 cells, under the same differentiating conditions, the c-abl RNA level did not change significantly, showing that Arg and c-abl responded in a different way to the inducers of differentiation used.

PML/RAR alpha transcripts monitored by polymerase chain reaction in acute promyelocytic leukemia during complete remission, relapse and after bone marrow transplantation.

The translocation t(15;17)(q24;q21), unique to acute promyelocytic leukemia (APL), gives rise to PML/RAR alpha fusion transcripts detected by the sensitive reverse transcriptase-polymerase chain reaction (PCR) technique. PCR may help in the diagnosis and in monitoring minimal residual disease. Reversion of PCR to negative is obtained by chemotherapy (CT) alone or in combination with all-trans retinoic acid (ATRA). Here we show a serial PCR study of 10 APL cases. Five cases were studied at the time of diagnosis, and all were PCR positive for the rearranged transcripts (three bcr1 type, two bcr3 type). Seven cases in complete remission (CR) after one cycle of induction CT were persistently PCR negative, one case in CR after ATRA rescue was persistently PCR positive (bcr1 type), one patient (bcr3 type) relapsed 15 months after the PCR-negative CR and one patient died early. Seven patients underwent bone marrow transplantation (BMT) (five allogeneic, two autologous). One of them died early after take of the allogeneic BMT, the other six cases studied by serial PCR were persistently negative. At a median follow-up of 31 months (range 9-39), none of these six cases had relapsed. PCR data characterize the CR at the molecular level and evaluate the efficacy of different treatments, including BMT. The data may help to define a standardized schedule for PCR follow-up, and are also potentially useful to establish the time required before judging patients with persistently negative PCR to be cured. BMT as post-induction treatment in first CR is also discussed.

Detection by polymerase chain reaction of BCR/ABL transcripts in myeloproliferative diseases at time of diagnosis and for monitoring chronic myelogenous leukaemia patients after bone marrow transplantation.

The Philadelphia chromosome t(9;22)(q34;q11) is a cytogenetic marker for chronic myelogenous leukaemia (CML), and is also present in some acute leukaemias. The translocation in CML gives rise to two BCR/ABL chimeric transcripts (b3a2 and b2a2) encoding a 210-kD tyrosine kinase protein. These leukaemia-specific transcripts can be detected easily by the reverse transcriptase polymerase chain reaction (PCR). PCR has improved the possibility of detecting minimal residual leukaemia cells in Ph-positive patients, especially after bone marrow transplantation (BMT). With PCR, we looked for BCR/ABL transcripts in 30 patients with CML and 4 with essential thrombocythaemia at time of diagnosis, finding a significant difference in the platelet counts of CML patients carrying b3a2 or b2a2 transcripts. The BCR/ABL transcript was monitored by PCR in 6 CML patients after BMT. The usefulness of PCR in clinical practice at time of diagnosis, and the biological and clinical significance of positive/negative PCR results, in patients with transplants, are discussed.

Polypeptide composition of the 8S form of prolyl-tRNA synthetase from rat liver.

Rat liver Fraction X containing the 24S complex of nine aminoacyl-tRNA synthetases, including prolyl-tRNA synthetase, was centrifuged on a 15-35% sucrose density gradient to obtain the 8S form of prolyl-tRNA synthetase. The enzyme was purified on a prolyldiaminohexyl-Sepharose 4B affinity column, specifically binding prolyl-tRNA synthetase to Sepharose-bound proline. After SDS-polyacrylamide gel electrophoresis, two peptides of 58 and 61 kDa were detected in the peak of prolyl-tRNA synthetase activity eluted from the affinity column. The 58 and 61 kDa peptides were also present in the 24S complex containing prolyl-tRNA synthetase activity isolated on the sucrose density gradient.

ATP- and ATP+ ubiquitin-stimulated proteolysis in rat liver and Yoshida ascites hepatoma.

To gain insight into the activity of cytosolic proteases in tumors, the ATP-dependent proteolysis of cell sap and the ATP- and ubiquitin-dependent proteolysis of Fraction II (a cytosolic subfraction freed of endogenous ubiquitin) were measured in the anaplastic Yoshida ascites hepatoma AH 130. Hepatoma cell sap showed only low, although significant, ATP-stimulated proteolysis, as best seen by comparisons with rat liver made on the basis of wet weight. Much of the basal proteolytic activity of cell sap and of its subfraction enriched in high Mr complexes (Fraction X) peaked near 18S in sucrose gradients. In contrast with cell sap, Fraction II from hepatoma degraded [14C]methylcasein more efficiently than Fraction II from normal liver, but the activities for liver and tumor did not differ on a wet weight basis. Altered polypeptide patterns shown by SDS-PAGE in the Yoshida hepatoma suggested that some abundant hepatoma-specific cytosolic protein might interfere with degradation of the [14C]methylcasein by hepatoma.

Effects of liver regeneration on tRNA contents and aminoacyl-tRNA synthetase activities and sedimentation patterns.

The tRNA content and aminoacyl-tRNA synthetases of regenerating liver in the phase of rapid growth were compared with those of livers from both intact and sham-operated rats. At 48 h after hepatectomy, the amount of active tRNA (called 'total acceptor capacity') is significantly higher in regenerating liver than in control livers, owing to a general, possibly not uniform, increase in the various tRNA families, which suggests that it may contribute to the increased protein synthesis and to decreased protein degradation as well. The activities of most, but not of all, aminoacyl-tRNA synthetases in cell sap of regenerating liver tend to be greater than normal. Increased activity of histidyl-tRNA synthetase fits in with the possibility that the mechanisms that control the rate of protein degradation through aminoacylation of tRNAHis in cultured cells [Scornik (1983) J. Biol. Chem. 258, 882-886] also operate in the liver and play a role in regeneration. Sedimentation analysis of cell sap in sucrose density gradients shows a shift of prolyl-tRNA synthetase activity toward the high-Mr form in regenerating liver. This change might be related to the positive protein balance and to growth in vivo, since it is also observed in the anaplastic Yoshida ascites hepatoma AH 130.

[Multivariate analysis of aminoacyl-tRNA synthetase activities of liver and hepatomas (author's transl)]. / Analisi multivariata delle attività aminoacil-tRNA sintetasiche del fegato e degli epatomi

Data on the aminoacyl-tRNA synthetase activities in normal liver and in three transplantable rat hepatomas, viz Yoshida's AH 130 and Morris's 5123C and 7793, were studied by means of factor analysis, a powerful technique of multivariate analysis particularly suitable foridentifying factors which theoretically may account for the pattern of interrelations between several variables. The analysis has been performed on 51 patterns of enzyme activities, covering 17 out of the 20 aminoacyl-tRNA synthetases; 80% on average of the total variability of the 17 enzyme activities may be accounted for by the first 4 factors extracted. The enzymes seem to fall into different groups, depending on their relationship with the factors thus identified. The results suggest that enzymes belonging to the same group share a common control mechanism, and are independent of the enzymes belonging to different groups, both in normal liver and in hepatomas.