Density functional theory based screening of ternary alkali-transition metal borohydrides: a computational material design project.

We present a computational screening study of ternary metal borohydrides for reversible hydrogen storage based on density functional theory. We investigate the stability and decomposition of alloys containing 1 alkali metal atom, Li, Na, or K (M(1)); and 1 alkali, alkaline earth or 3d/4d transition metal atom (M(2)) plus two to five (BH(4))(-) groups, i.e., M(1)M(2)(BH(4))(2-5), using a number of model structures with trigonal, tetrahedral, octahedral, and free coordination of the metal borohydride complexes. Of the over 700 investigated structures, about 20 were predicted to form potentially stable alloys with promising decomposition energies. The M(1)(Al/Mn/Fe)(BH(4))(4), (Li/Na)Zn(BH(4))(3), and (Na/K)(Ni/Co)(BH(4))(3) alloys are found to be the most promising, followed by selected M(1)(Nb/Rh)(BH(4))(4) alloys.

Temporal variation in the expression of the p53 protein in proliferating and differentiating murine erythroleukaemia cells.

Temporal changes in the expression of p53 were investigated during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukaemic (MEL) cells. Cell preparations were analysed by SDS-PAGE and western immunoblotting, which detected an immunospecific band of molecular mass 53 kDa. This analysis provided semi-quantitative information. Cell extracts were analysed further by means of ELISA techniques, using a p53-specific antibody, to provide quantitative data. In time series experiments in which cells were isolated at 15, 30 and 60 min intervals, dynamic variations in the expression of the p53 protein were detected in both the untreated and the HMBA-treated MEL cells. In all cases, the effects were complex with variations in amplitude, frequency and phasing of the rhythms. The available evidence suggests that the observed patterns, like periodic variations in other systems, are modified in rhythmic fashion with respect to period and amplitude. The results add further support for our view that it is essential to consider the dynamics when interpreting the role of p53 and cellular processes in general.

Temporal changes in the expression of protein phosphatase 1 and protein phosphatase 2A in proliferating and differentiating murine erythroleukaemia cells.

Rhythmic changes in the expression of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) were investigated during hexamethylene bisacetamide (HMBA) induced differentiation of murine erythroleukaemic (MEL) cells. Cell extracts were analysed by SDS-PAGE and western immunoblotting using specific antibodies. An immunospecific band of molecular mass 36 kDa (catalytic subunit) was detected for PP1. For PP2A, two immunospecific bands of 32 kDa (proteolytically cleaved catalytic subunit) and 36 kDa (catalytic subunit) were observed. Comparisons of proliferating and differentiating cells using only one time point showed no significant differences between mean values for the expression of the PP1 or PP2A enzyme proteins. This kind of analysis, implying that HMBA had little effect, proved misleading, as comparisons using multiple time points showed rhythmic patterns of protein expression which were modulated by the differentiating agent. The effects were complex affecting both the frequency and phasing of rhythms. The results add further support for the view that live cells are multi-oscillators and for the concept that differentiation depends on changes in temporal organization of complex autodynamic feedback loops and multiple interactions between control circuits performing in parallel. In particular, modulation of the dynamics of key proteins, such as PP1 and PP2A, may be a possible mechanism for controlling cellular function and reversing transformation in accordance with long standing theoretical and other experimental data.

Temporal variations in protein tyrosine kinase activity in leukaemic cells: response to all-trans retinoic acid.

Protein tyrosine kinases (PTKs) play a critical role in the modulation of a wide variety of cellular events such as cell division, differentiation and metabolism. Regulation of PTK activity must be tightly controlled as over-stimulation is known to impair normal cell growth, resulting in oncogenic transformation. Since evidence suggests that dynamic oscillatory behaviour occurs in metabolic control processes, we investigated the patterns of oscillatory behaviour in the total protein content and enzyme activity of PTK exhibited by proliferating and differentiating human acute promyelocytic cells. Distinct rhythmic patterns of oscillatory behaviour were observed in both the amount of extractable protein and PTK enzyme activity. Rhythmic characteristics such as period and amplitude were significantly modulated following treatment with all-trans retinoic acid, an inducing agent. These results support the view that dynamic oscillatory control processes may play an important role in regulating cellular behaviour.

Modification of oscillatory behaviour of protein tyrosine kinase and phosphatase during all-trans retinoic acid-induced differentiation of leukaemic cells.

Granulocytic maturation of human acute promyelocytic leukaemic (HL-60) cells was induced using all-trans retinoic acid (ATRA). Time-dependent changes in the enzyme activities of protein tyrosine phosphatase (PTP) and protein tyrosine kinase (PTK), and the total extractable protein content were monitored in proliferating and differentiating cells. The existence of periodicity was demonstrated clearly in both PTP and PTK enzyme activities and in the amount of protein extracted from the cells. Following ATRA treatment, differentiation-induced changes in rhythmic characteristics such as period and amplitude were evident. A noticeable effect was that of ATRA on the enzyme activity of PTP, for which four distinct patterns of oscillatory behaviour were identified. This study examines these changes, in an attempt to gain insight into the role which biochemical oscillators may play in the regulation of molecular control mechanisms.

Rhythmic patterns in the expression of the ras oncogene in proliferating and differentiating erythroleukaemia cells.

Temporal variations in the expression of the ras oncogene, and its protein product, were investigated during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukaemic (MEL) cells. We highlight the fact that when comparisons were made between untreated, proliferating cells and HMBA-treated, differentiating cells using only one time-point, differences, both for the expression of the gene and the protein, were in most cases insignificant; standard deviations were high and the interpretation could be made that HMBA had little effect. Such interpretation fails to take account of the dynamic nature of the system, with single time-point studies giving incomplete information, which can be misleading. Multiple time analyses showed clearly that rhythmic patterns of expression were modulated by the differentiating agent. Time-dependent changes in the expression of mRNA specific to H- ras and N- ras, as well as in the expression of the Ras protein, when measured over periods of minutes or hours, were apparent. HMBA affected frequency and phasing of the rhythms. Regulation of the dynamics in this way may be crucial to the control of cell function and transformation.

Protein kinase c in erythroleukaemia cells: temporal variations in the expression of the alpha, epsilon and zeta isoforms.

Determinations of mRNA and enzyme protein analyses for the protein kinase C (PKC) isoforms, alpha, epsilon and zeta, representing the classical, novel and atypical groups, respectively, in murine erythroleukaemic (MEL) cells revealed the occurrence of cyclic behaviour in expression. Such rhythmicity was not apparent when multiple analyses using only a single time-point were considered; the importance of incorporating a time component into experimental design was demonstrated. On induction of differentiation over a period of five days with hexamethylene bisacetamide, changes in the patterns of expression of mRNA and protein were apparent, as compared with those of untreated, proliferating cells. Modulation of the dynamics of the various PKC isoforms may represent a regulatory mechanism for the control of a diversity of cellular functions.

Temporal variations in protein tyrosine phosphatase activity during cell proliferation and differentiation.

Cellular oscillations in total extractable protein content and protein tyrosine phosphatase activity were analysed in proliferating and differentiating human acute promyelocytic leukaemic (HL-60) cell extracts. Differentiation was induced along the granulocytic pathway using all-trans retinoic acid (ATRA). High frequency rhythms with distinct, well defined waveforms of varying amplitude were observed for both the total protein content and for the enzyme activity of protein tyrosine phosphatase (PTP). Linear correlation analysis showed that there was no obvious relationship between the protein content and the corresponding PTP activity, suggesting that the periodic variations between these two components are relatively independent of each other. ATRA significantly altered the characteristics of the rhythms with respect to the period and amplitude and had a dampening effect on the oscillatory pattern of PTP activity. Modulation of such characteristics may be of significance with respect to the regulation of the differentiation processes and the possible reversal of transformation.

Transient chaos in intracellular dynamics?

Periodogram analyses of the temporal variations of several cellular oscillations occasionally reveal the existence of short duration bands containing a wide range of frequencies. The possibility is considered that these are due to the transient compliance with chaotic conditions.

High frequency oscillations in the activity of phosphotyrosine phosphatase in murine erythroleukaemic cells: action of insulin and hexamethylene bisacetamide.

We have previously reported oscillations in the activities of the phosphoamino acid phosphatases in murine erythroleukaemic cells. In keeping with our predictions we now show that the phosphotyrosine phosphatase activity rhythm has a much shorter period than originally seemed the case, being of the order of 10 min and probably less. The periodic changes show evidence of rhythmic modulation of mean, period and amplitude as with all other cellular oscillations studied. Insulin decreases the frequency of the rhythm while the inducer of differentiation, hexamethylene bisacetamide (HMBA) decreases its amplitude. Current ideas on phosphorylation dynamics in relation to metabolism and mitosis may need to be revised in the light of the observations.

Independent high-frequency oscillations in the amounts of individual isozymes of lactate dehydrogenase in HL60 cells.

Early studies, using kinetic methods, suggested that the isozyme pattern of lactate dehydrogenase in various cells oscillated with time. More recent electrophoretic studies on murine erythroleukaemic cells (which exhibit only one isozyme) indicated very high frequency variations (period 2 min or less) in the amount of the lone active isozyme. We now show that in HL60 cells, the activity stain intensities of the two major isozyme bands both oscillate but the temporal variations are distinct. As with other cellular rhythms, each of the two periodicities seem to be modulated in cyclic fashion with respect to period, amplitude and mean levels, the periods of both the primary and modulating rhythms being of the order of 10-15 min or probably much less.

Distinct, very high frequency oscillations in the activity and amount of active isozyme of lactate dehydrogenase in murine erythroleukaemic cells and a cell-free system.

Electrophoretic and kinetic determinations of the activity of lactate dehydrogenase in murine erythroleukaemic (MEL) cells, sampled at 1 min intervals, reveal distinct oscillations in the activity and amount of active isozyme. Both oscillations have periods in the range of 2-6 min (probably less) and both appear to be rhythmically modulated with respect to period, amplitude and mean. The oscillations also occur in cell-free systems, a fact which throws doubt on the value of studies where it is assumed that such preparations have constant composition.

Effect of tumour promoting agents on protein phosphorylation in human placenta.

1. The effects of okadaic acid (OA) and phorbol-12-myristate-13-acetate (PMA) on protein phosphorylation were studied in human term placentas. 2. When samples treated with tumour promoters were compared with untreated samples, the phosphorylation of a 135 kDa protein was significantly decreased; OA also produced a decrease in phosphorylation of a 24 kDa protein. 3. Both substances produced an alteration in the proportions of bands of masses 170, 65 and 24 kDa, relative to total phosphorylation; PMA treatment also affected the band of mass 135 kDa. 4. Placental cell extracts were also subjected to Western blotting with a protein kinase C (PKC) antibody, reportedly specific for the alpha- and beta-isoforms. 5. Two immunoreactive proteins were detected; an 80 kDa band, presumably corresponding to the alpha- or beta-PKC, and a 64 kDa protein, which could be a degradation production of the 80 kDa protein or it could correspond to another form of the enzyme. The expression of PKC did not change on treatment with PMA.

Insulin stimulation of high frequency phosphorylation dynamics in murine erythroleukemia cells.

The phosphorylation potentials of two proteins (M(r) 81 kDa and 63 kDa) in extracts of murine erythroleukemic (MEL) cells both vary in an oscillatory manner, sometimes changing by as much as 100-fold in 10 min. Direct analysis of the temporal changes indicates the existence of periodic modulation of the frequencies, amplitudes and mean levels of the two rhythms. In both cases, periodogram analyses, by two methods, confirm the presence of several oscillations having periods in the range 20-100 min which tend to occur in (pseudo) periodic bursts. Insulin has been found to enhance these oscillations in a manner comparable with its effect on rhythmic variations in cell morphology. Despite the marked similarity in the behaviour of the two proteins, no particular phase relationship existed between the two temporal variations, suggesting differences between the underlying driving forces.

Oscillatory variations in the amount of protein extractable from murine erythroleukemia cells: stimulation by insulin.

The amount of protein extractable from murine erythroleukemia (MEL) cells varies in an oscillatory manner at high frequency and high amplitude as it does for several other cell lines. Moreover, the rhythm appears to be modulated in periodic fashion with respect to the mean, period and amplitude. The phenomenon thus seems to be universal and fundamental. Time series analyses support the view that several periodicities contribute to the observed protein rhythm. Insulin affects the dynamics as it does for both morphological and phosphorylation oscillations. Caution is necessary in the interpretation of 'specific activities' of cellular components and in electrophoretic studies wherein equal amounts of protein are applied to the wells in an effort to correct for random errors.

Glucocorticoid receptor binding in proliferating and differentiating cells: modulation by a tumour promoting agent.

The effects of the tumour-promoting agent, phorbol 12-myristate 13-acetate, on dexamethasone binding and protein kinase C in proliferating and differentiating murine erythroleukemic cells were investigated. Concentration dependent changes in steroid binding were observed in proliferating and differentiating cells treated with phorbol 12-myristate 13-acetate for 2h or 24h. Ligand binding in differentiating cells was consistently lower than in proliferating cells. Reduced expression of protein kinase C was observed in differentiating cells as compared with proliferating cells; the amount of enzyme protein was reduced on treatment of cells with phorbol ester.

Phosphorylation of endogenous protein in primate kidney: effects of modulators of calcium/calmodulin-dependent kinases.

1. Phosphorylation of endogenous proteins in response to calcium and calmodulin was assessed in membrane and cytosol from primate kidney. 2. Quantitative studies showed that calcium and calmodulin had little effect on phosphorylation in baboon kidney cytosol; in membranes, phosphorylation was significantly decreased by calcium and calmodulin. 3. Phosphorylation of specific proteins which had been electrophoretically separated indicated that calmodulin, in the absence or presence of calcium, enhanced bands of masses 37.1 +/- 1.1 kDa and 103.3 +/- 6.2 kDa (N = 6) in cytosol fractions of baboon kidney; in membrane fractions no effect was noted. 4. Similar results were found in normal human kidney.

Phosphorylation of endogenous protein in primate kidney. Effects of cyclic AMP.

1. Phosphorylation of endogenous proteins in response to cyclic AMP was assessed in membrane and cytosol from primate kidney. 2. Quantitative studies showed that cAMP significantly increased phosphorylation in baboon kidney membranes; in cytosol there was no effect. 3. Phosphorylation of specific proteins which had been electrophoretically separated showed that five major bands were intensified by cAMP in baboon membranes; in cytosol, three bands were intensified. Similar results were found in normal human kidney. 4. Photoaffinity labelling indicated that a 56 kDa band phosphorylated in cytosol may correspond to the regulatory subunit of type II cAMP-dependent protein kinase.

Phosphoamino acid phosphatases in normal and cancerous tissues of the human uterus, cervix and ovary.

The activities of phosphoamino acid phosphatases were measured in human myometrium and fibroma and normal and cancerous tissues of the cervix and ovary. Phosphoserine and phosphothreonine phosphatases were detected only in myometrium and fibroma and the values were relatively low. Phosphotyrosine phosphatase activities in myometrium and fibroma fell within a similar range; this was also the case for ovary and ovarian carcinoma, whereas values for cervical tumours were significantly higher than for normal cervix. Activities of phosphotyrosine phosphatase in serum from patients with cervical or other tumours were, in most cases, within the range of values obtained for normal serum.

Subcellular fractionation of murine erythroleukemic cells: distribution of protein kinases.

A method for subcellular fractionation of murine erythroleukemic cells is described; a highly purified cytosol fraction and significantly enriched membrane, mitochondrial, and nuclear fractions were obtained. During development of the procedure, we demonstrated how the composition of the extraction buffers and the techniques used can affect activity and distribution of protein kinases. Protein kinases in the various fractions were separated by nondenaturing electrophoresis and detected by phosphorylation and autoradiography. Differences in the relative proportions of the kinases, which may be significant in relation to differentiation, were seen in all the fractions on hexamethylenebisacetamide treatment of the cells.