Diversity of peripheral blood mononuclear cells as revealed by a novel multiple microgel "comet assay".

Multiple microgel comet assay (MMCA) is a metho-dological adaptation of the single-cell gel electrophoresis assay in which we have introduced the use of standard agarose plug molds in an attempt to improve and expand the applications of the assay. We focused on the study of the heterogeneity of peripheral blood mononuclear cells (PBMC) at the level of the basal single-strand breakage and the DNA damage induction caused by ionizing radiation. Differences among subpopulations were also investigated at the level of chromatin organization and methylation after NotI digestion of microgel-embedded cells. In parallel experiments, the NotI-digested nucleoids were also analyzed with the use of pulsed-field gel electrophoresis (PFGE) and the DNA migration patterns were compared with the corresponding patterns from the MMCA. Significant heterogeneity in the distribution of the oxidative DNA damage, as well as intracellular variations in the NotI digestion patterns were observed in the cell population of PBMC. The combined use of both the comet assay and PFGE provides a useful model for analysis of variation in DNA damage in individual cells as well as information on size of DNA fragments.

Elevated levels of the chromosomal protein HMG 17 in chronic myelogenic leukemia.

The High Mobility Group protein HMG 17 has been isolated from human leukemia cells obtained from patients with chronic myelogenic leukemia (CML). The level of expression of HMG 17 was investigated Human leukemia cells have three times more HMG 17 than normal human leukocytes. Three other malignant tissues were also compared. Two of these breast adenocarcinoma and other intestine- also exhibit higher amounts of HMG 17. The elevated expression of HMG 17 suggests that the level of the protein may be associated with rates of cellular proliferation.

The effect of chlorambucil on the biosynthesis of the HMG and histone H1 chromosomal proteins of HEp-2 cells.

The effect of chlorambucil, a bisalkylating agent, on the biosynthesis of the 5% PCA extractable protein fraction of the cancer cell line, HEp-2, has been analyzed. It was found that the synthesis of all the high mobility group proteins as well as that of the H1 and H1o histone proteins are inhibited by this agent. HMG 14 and the H1, H1o proteins are inhibited to the same extent as that reported for the core histones of the same cell line [7], while slightly higher levels of inhibition were found for the HMG 1, 2 and 17 proteins. The proteins, P1 and HMG I exhibited the highest level of inhibition of the entire fraction. These findings extend previous findings regarding the histone proteins and may be correlated to a dysfunction in the normal process of chromatin condensation and a potential cytotoxic effect of this agent during the G2 phase.

Experimental transplantation of hepatocytes in cases of toxic acute liver failure. An allograft model.

The aim of this experimental study was to modify a rat liver-cell harvesting technique and to evaluate the efficacy of allogeneic liver cell transplantation (Tx) using cyclosporin A immunosuppression in rats with N-dimethylonitrosamine (N-DMNA)-induced acute liver failure (ALF). Twenty male Wistar rats, weighing 190-320 g, were used as donors. Hepatocytes were harvested by the use of a modification of the Seglen portal vein collagenase perfusion technique (type V/1.3 mg/ml), which resulted in the isolation of a mean of 8000 viable clusters of hepatocytes per donor (viability was measured using the trypan blue exclusion test). The male Lewis recipients and controls received 20 mg/kg N-DMNA i.v., and were then divided in three groups. Group 1 (n = 5) received no treatment, group 2 (n = 10) received 5000 clusters of freshly isolated hepatocytes (FIH) in the spleen 24 h after the administration of N-DMNA- and group 3 (n = 10) received 5000 clusters of FIH beneath the renal capsule, 24 h after the administration of N-DMNA. All groups were treated with cyclosporin A 20 mg/kg per day i.p.. SGOT and bilirubin values were measured and all surviving rats were sacrificed on day 14. All rats in group 1 died of histologically confirmed liver necrosis within 72 h. The 14-day survival was 60% in group 2 and 50% in group 3. The post-Tx SGOT values reached their maximum on days 3-4 (group 2, mean 754; group 3, mean 529) and were only slightly elevated on day 14 (group 2 = 75, group 3 = 48). The post-Tx bilirubin values reached their maximum on days 3-5 (group 2 = 1.1, group 3 = 1) but failed to return to normal until day 14. Autopsy and histological examination of the surviving animals showed well-preserved hepatocellular spherical aggregates in the spleen and hepatocellular "cords" in the kidney accompanied by signs of regeneration of the native liver. We concluded that the hepatocyte Tx in a rat experimental allo-Tx model improved the survival rate and the SGOT values in cases of toxic ALF. Survival rates between the two different sites of Tx were similar.

Combined hepatocyte-islet transplantation: an allograft model.

Experimental hepatocyte transplantation (Tx) has been shown to improve the survival rate of acute hepatic failure (AHF) in different models. Histological and biochemical data from some studies suggest more satisfactory function of hepatocytes after combined hepatocyte-islet Tx. The aim of the present study was to compare the survival rate between two different sites of Tx (kidney subcapsular and spleen) of hepatocytes alone or combined with islets of Langerhans in rats with surgically induced AHF (90% hepatectomy) accross a major histocompatibility barrier (WAG to Lewis). Rats were divided into five groups (n = 6 in each group). Group 1 consisted of AHF without treatment, group 2, AHF followed by hepatocyte Tx into the spleen, group 3, AHF followed by hepatocyte Tx subcapsular into the kidney, group 4, AHF followed by combined hepatocyte islet Tx into the spleen, and group 5, AHF followed by combined hepatocyte islet Tx subcapsular into the kidney. The number of hepatocytes was 10(7) and the number of islets was 400. All rats received cyclosporin A (CsA) i.v. (20 mg/kg on days 0-4 and 10 mg/kg on days 5-30). Hepatocytes were harvested using a modification of the portal vein collagenase perfusion (type V 1.3 mg/ml) and islets, with the collagenase digestive technique (type XI 1 mg/ml). All Tx took place 24 h after AHF. All rats in group 1 died within 48 h. In groups 2 and 3, the combined survival rate was 33% 1 month after Tx, while in groups 4 and 5, the combined survival rate was 50% at 1 month. All surviving animals were sacrificed and histological examination showed well-preserved hepatocellular aggregates in the spleen and beneath the renal capsule, as well as islets around the clusters of hepatocytes. SGOT and SGPT values were also measured. We concluded that the combined Tx in a rat experimental allo-Tx model in cases of AHF improves the survival rate in comparison with hepatocyte Tx alone. The survival rate at the two different sites for combined Tx was similar.

Influence of chlorambucil, a bifunctional alkylating agent, on the histone variant biosynthesis of HEp-2 cells.

The effect of chlorambucil on the synthesis of histone variants of a cancer cell line HEp-2 is analysed and compared to that of nontreated and hydroxyurea treated cells. Cell proteins were labelled with [14C]lysine and [14C]arginine and histone variants resolved by one- or two-dimensional electrophoresis. Chlorambucil shows no significant decrease in total protein synthesis but shows a significant decrease in histone biosynthesis. It does not selectively inhibit the synthesis of the S-phase variants, i.e., H2A.1, H2A.2, H3.2 or the G1/G2 phase (basal) histone variants, i.e., H2A.Z, H2A.X and H3.3. On the contrary, hydroxyurea treated cells, which also show no significant decrease in amino acid incorporation into total cellular protein but do exhibit a significant inhibition of histone biosynthesis, show a selective inhibition of the synthesis of S-phase variants, but have no effect on the synthesis of basal histone variants. On the basis of histone variants being synthesized in the presence of chlorambucil, it is shown that although chlorambucil shows a specificity for histone synthesis inhibition it has a general action over the whole variant complement and is not coupled to S-phase synthesis in a way typical for DNA synthesis inhibiting drugs.

Histone variants of the insect Plodia interpunctella during metamorphosis.

The pattern of histone variants from the meal moth Plodia interpunctella was compared to the mouse histone variant pattern. Plodia contains histones which comigrate on two dimensional gels with H3.2, H3.3, H4 and H2A.Z in mouse. Plodia H2A.1 and H2B.1 migrate somewhat differently from the respective mouse histones. Comparison of the iodinated tryptic peptides of H2A.1 and H2A.Z from mouse and Plodia showed that the H2A.Z proteins have two iodinated peptides that comigrate in the two species and three more that are different. The H2A.1 proteins in the two species have one iodinated peptide which comigrates and two more which migrate very close to each other. The histone variants from three developmental stages, larval, pupal and adult of Plodia interpunctella were also identified and compared. The same histone variant pattern is found through all stages of development. It is concluded that histone gene expression does not change during metamorphosis in Plodia .