The accessory subunit of mitochondrial DNA polymerase gamma determines the DNA content of mitochondrial nucleoids in human cultured cells.

The accessory subunit of mitochondrial DNA polymerase gamma, POLGbeta, functions as a processivity factor in vitro. Here we show POLGbeta has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGbeta increased nucleoid numbers, whereas over-expression of POLGbeta reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGbeta altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGbeta preferentially bound to plasmids with a short displacement-loop, in contrast to POLGalpha. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGbeta is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.

The combination of the antitumoural pyridyl cyanoguanidine CHS 828 and etoposide in vitro--from cytotoxic synergy to complete inhibition of apoptosis.

1. The present study was aimed at elucidating the apoptosis inhibitory properties of the cyanoguanidine CHS 828. CHS 828 exhibits impressive cytotoxic activity in vitro and in vivo. Apoptosis is not its main mode of cytotoxic effect, and we have previously proposed a dual mechanism, where CHS 828 inhibits its own cell death pathways. 2. Etoposide on the other hand, is a well-established anticancer agent with documented effect in a number of malignancies, induces apoptosis through extensively studied caspase dependent pathways. 3. Here we studied the combined effect of the two drugs in the human lymphoma cell line U-937 GTB. Cytotoxicity was evaluated as total viability measured by the fluorometric microculture cytotoxicity assay (FMCA). Caspase activity was assessed by colorimetric detection of specific cleavage products for caspases 3, 8 and 9, respectively. Morphology was evaluated in May-Grünwald/Giemsa stained preparations. Interaction analysis based on FMCA results of simple combination exposure revealed impressive synergistic effect on cell kill. 4. Detailed investigations of the kinetics involved showed that short pre-exposure (0-12 h) to CHS 828 enhanced caspase activation by etoposide, while longer pre-exposure (18-48 h) inhibited both caspase activation and apoptotic morphology otherwise induced by etoposide. The present results support the theory that CHS 828 block specific cell death pathways. 5. The synergistic results are promising for future combination trials in animals, however, different dosing schedules should be considered, in order to investigate whether the above findings translate into the in vivo setting.

Characteristics of etoposide-induced apoptotic cell death in the U-937 human lymphoma cell line.

Cell death induced by etoposide in the human lymphoma cell line U-937 GTB was characterized. Activity of caspases -3, -8 and -9 was measured by spectrophotometric detection of specific cleavage products, DNA fragmentation by TdT-mediated dUTP nick end-labelling (TUNEL), and apoptotic morphology by conventional staining and microscopy, as well as by a novel method-the microculture kinetics (MiCK) assay. Synthesis of protein and DNA during exposure was monitored by incorporation of radioactive leucine and thymidine, respectively. The effects of caspase inhibitors on total viability, as well as early and late morphological changes were studied. Etoposide rapidly induced apoptosis, dependent on caspase-3 and -8, but inhibition of these caspases did not prevent major cell death, but promoted a switch in late morphology. The novel MiCK assay added valuable information on early morphological events during cell death. Hence, this study provides support for caspase-8-mediated apoptosis in U-937 GTB when exposed to etoposide. General caspase inhibition switches cell death to one with a different morphology.

Kinetics of charge separation and A0- --> A1 electron transfer in photosystem I reaction centers.

The charge separation P700*A(0) --> P700(+)A(0)(-) and the subsequent electron transfer from the primary to secondary electron acceptor have been studied by subtracting absorption difference profiles for cyanobacterial photosystem I (PS I) complexes with open and closed reaction centers. Samples were excited at 660 nm, which lies toward the blue edge of the core antenna absorption spectrum. The resulting PS I kinetics were analyzed in terms of the relevant P700, P700(+), A(0), and A(0)(-) absorption spectra. In our kinetic model, the radical pair P700(+)A(0)(-) forms with 1.3 ps rise kinetics after creation of electronically excited P700*. The formation of A(1)(-) via electron transfer from A(0)(-) requires approximately 13 ps. The kinetics of the latter step are appreciably faster than previously estimated by other groups (20--50 ps).

Cell death with atypical features induced by the novel antitumoral drug CHS 828, in human U-937 GTB cells.

N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828), with promising antitumoral effects in vitro and in vivo, is currently in clinical Phase I and II studies. Its exact mechanism of action is unclear, but previous studies indicate that CHS 828 induces a controlled, delayed mode of cell death. The characteristics of the cell death process were investigated in vitro in the apoptosis-prone cell line U-937 GTB. Mitochondria showed hyperpolarization at 24 to 32 h and a subsequent late disruption of mitochondria membrane potential (Deltapsi(m)). Between 44 and 72 h of CHS 828 exposure, there was an increasing frequency of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) positive cells indicative of apoptosis, but caspase-3 was only modestly increased and caspases-8 and -9 showed no activation upon CHS 828 exposure. Furthermore, the morphology of exposed cells did not conform to classical apoptosis, and viability and morphology were unaffected by inhibition of caspases. Thus, CHS 828 induces several unexpected features in this system, suggesting a potentially novel mechanism of action.

Temporal effects of the novel antitumour pyridyl cyanoguanidine (CHS 828) on human lymphoma cells.

CHS 828, a novel pyridyl cyanoguanidine, has shown potent antitumour activity both in vitro and in vivo and is currently undergoing phase I evaluation in humans in collaboration with the European Organization for Research and Treatment of Cancer (EORTC). Here we study the temporal effects of CHS 828 on cytotoxicity, protein and DNA synthesis, cellular morphology and ultra structure using the lymphoma cell line U-937 GTB as the primary tumour model. In vitro analysis of tumour cell survival in response to CHS 828 revealed a cytotoxic effect progressively increased as a function of exposure time with maximum efficacy observed after 72 h. Activity of CHS 828 on U-937 GTB cells grown in vivo was also found. CHS 828 induced-cell death was dependent on intact protein synthesis and most cells appeared to lose their membrane integrity in the presence of a relatively well preserved nuclear structure. The results indicate that CHS 828 induced active and delayed cell death with a non-apoptotic morphology.

Apoptosis induced by calcein acetoxymethyl ester in the human histiocytic lymphoma cell line U-937 GTB.

Effects of calcein acetoxymethyl ester (calcein/AM) on macromolecular synthesis, mitochondrial membrane potential, and mode of death were studied in U-937 GTB lymphoma cells. This was accomplished by measurements of (14)C-labeled thymidine and leucine incorporation, 5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) and caspase-3 activity measurements, TdT-mediated dUTP nick end labeling (TUNEL) staining, morphology, and a newly developed assay of apoptosis detection, the microculture kinetic assay (MiCK). This assay, based on absorbance measurements of cells, has been reported to reflect morphological changes in apoptosis. At 2.5 microg/mL, rapid inhibition of DNA and protein synthesis resembling that of the known inhibitors, aphidicholin and cycloheximide, was observed. Decreased mitochondrial membrane potential was evident after 1 hr of exposure and was followed by an increase in caspase-3 activity, while at 6 hr 30% of cells appeared positive with TUNEL staining. After 12 hr of exposure, viability was less than 5% as judged by morphological examination. In the MiCK assay, calcein (2.5 microg/mL) gave a rapid rise in absorbance after 3.5 hr of exposure with a peak at 5 hr, indicating maximum extent of apoptosis at that time. This was similar to the pattern generated for etoposide and doxorubicin. The results indicate that calcein, similar to cytotoxic drugs, induces a strong apoptotic response within hours of exposure.

Induction and suppression of collagen-induced arthritis is dependent on distinct fcgamma receptors.

Receptors for immunoglobulin (Ig)G (FcgammaRs) are important for the antibody-mediated effector functions of the immune system. FcgammaRI and FcgammaRIII trigger cell activation through a common gamma chain, whereas FcgammaRII acts as a negative regulator of antibody production and immune complex-triggered activation. Here we describe the in vivo consequences of FcgammaR deficiency in a mouse model of human rheumatoid arthritis. FcRgamma chain-deficient mice on arthritis-susceptible DBA/1 background were immunized with collagen for induction of collagen-induced arthritis. The DBA/1 mice lacking FcRgamma chain were protected from collagen-induced arthritis in contrast to wild-type mice, although both groups produced similar levels of IgG anticollagen antibodies. In comparison, DBA/1 mice lacking FcgammaRII developed an augmented IgG anticollagen response and arthritis. These observations suggest a crucial role of FcgammaRI and FcgammaRIII in triggering autoimmune arthritis.

B800-->B850 energy transfer mechanism in bacterial LH2 complexes investigated by B800 pigment exchange.

Femtosecond transient absorption measurements were performed on native and a series of reconstituted LH2 complexes from Rhodopseudomonas acidophila 10050 at room temperature. The reconstituted complexes contain chemically modified tetrapyrrole pigments in place of the native bacteriochlorophyll a-B800 molecules. The spectral characteristics of the modified pigments vary significantly, such that within the B800 binding sites the B800 Q(y) absorption maximum can be shifted incrementally from 800 to 670 nm. As the spectral overlap between the B800 and B850 Q(y) bands decreases, the rate of energy transfer (as determined by the time-dependent bleaching of the B850 absorption band) also decreases; the measured time constants range from 0.9 ps (bacteriochlorophyll a in the B800 sites, Q(y) absorption maximum at 800 nm) to 8.3 ps (chlorophyll a in the B800 sites, Q(y) absorption maximum at 670 nm). This correlation between energy transfer rate and spectral blue-shift of the B800 absorption band is in qualitative agreement with the trend predicted from Förster spectral overlap calculations, although the experimentally determined rates are approximately 5 times faster than those predicted by simulations. This discrepancy is attributed to an underestimation of the electronic coupling between the B800 and B850 molecules.

An ultrafast time-resolved anisotropy study of bacteriochlorophyll a in pyridine.

The transient absorption anisotropy spectrum of bacteriochlorophyll a (BChl a) in pyridine was measured in the wavelength interval 550-850 nm, 1 ps after optical excitation with a 792-nm femtosecond light pulse. In the wavelength region of Q(y) absorption and stimulated emission (775-825 nm), the anisotropy was found to be close to the theoretically expected value (0.4) for a two-level system. In the wavelength region 650-750 nm, where the transient absorption signal is dominated by excited state absorption, the anisotropy is reduced to approximately 0.18. Anisotropy kinetics were measured at several wavelengths and found to be constant within the time window 0-5 ps, showing that no internal dynamics of the BChl a molecule change the anisotropy on the time scale of tens of picoseconds.

Importance of CD23 for collagen-induced arthritis: delayed onset and reduced severity in CD23-deficient mice.

Increased expression of the low affinity receptor for IgE, FcepsilonRII/CD23 has been observed in rheumatoid arthritis. In view of this, we have investigated the expression and influence of CD23 in collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. CD23+ cells were analyzed in lymph nodes of DBA/1 mice immunized with bovine collagen type II (BCII) in CFA or with CFA only. The percentage of CD23+ lymph node cells was increased in both BCII/CFA- and CFA-immunized mice at 1, 3, and 7 wk after immunization compared with unimmunized mice, indicating a role for the adjuvant to trigger general inflammation and CD23 expression. To investigate the functional role of CD23 in CIA, CD23-deficient mice on the DBA/1 genetic background were studied. After immunization with BCII/CFA, these mice developed CIA with delayed onset and reduced severity compared with wild-type mice. These findings suggest that an increased number of CD23+ cells is part of an inflammatory response and that CD23 expression is of pathogenic importance in the arthritic process.

Cytotoxic activity of cyclosporin A and [3-keto-Bmt1]-[Val2]-cyclosporin (SDZ PSC 833) on tumour cells from patients with haematological malignancies.

OBJECTIVE AND METHOD: The fluorometric microculture cytotoxic assay was employed for characterisation of the cytotoxic effect of cyclosporin A (CsA) and its non-immunosuppressive analogue SDZ PSC 833, [3-keto-Bmt1]-[Val2]-cyclosporin (PSC) in tumour cells from patients with haematological or solid tumours. RESULTS: Tumour cells from patients with chronic lymphocytic leukemia (CLL) or non-Hodgkin's lymphoma (NHL) were found to be more sensitive to both drugs than those of tumour cells from patients with acute lymphocytic leukemia (ALL), acute myoblastic leukaemia (AML) and various solid tumours. There was a close correlation between the effects of the two drugs (correlation coefficient 0.71), but CsA was slightly more active than PSC in most diagnoses. No tumour cells sample showed sensitivity to PSC without also being sensitive to CsA. There was a moderate level of correlation between the activity pattern of CsA and doxorubicin (correlation coefficient 0.66), whereas the correlations with other cytostatics, such as vincristine, cytarabine and melphalan, were low (correlation coefficient -0.11 to 0.33). CONCLUSION: The results indicate that PSC shares the direct cytotoxic properties of CsA, but is slightly less potent. Clinical testing of the cytotoxic effect of these agents in haematological malignancies seems warranted and the apparent non-cross-resistance with standard agents makes cyclosporins a potentially useful adjunct to chemotherapy in those diagnoses.