Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae.

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.

Effects of interleukin-1 beta on human pulpal fibroblast proliferation and collagen synthesis.

The aim of this study was to investigate the effect of interleukin-1 beta (IL-1 beta) on pulpal fibroblast proliferation and collagen synthesis in vitro. Cell proliferation was assessed by [3H]thymidine uptake at 24, 48, and 72 h. These cells were compared with dermal fibroblasts under a range of conditions. IL-1 beta inhibited pulp cell proliferation, but this effect was decreased by the presence of indomethacin. In contrast, IL-1 beta stimulated dermal fibroblast proliferation in the absence and presence of indomethacin. Collagen synthesis was analyzed by incorporation of 5-[3H]proline into polypeptide chains. IL-1 beta stimulated synthesis of type I collagen both in the absence and presence of indomethacin. Results suggest that the inhibitory effect on pulp cell proliferation is dependent upon IL-1 beta-induced prostaglandin E2 synthesis and that IL-1 beta is a potent mediator of prostaglandin E2 synthesis in dental pulp. These observations imply a role of IL-1 beta in collagen synthesis, but not in the fibroblast proliferation phase of the healing process.

Hematopoietic-specific genes are not induced during in vitro differentiation of scl-null embryonic stem cells.

The helix-loop-helix transcription factor, scl, plays an essential role in hematopoietic development. Embryos in which the gene has been disrupted fail to develop yolk sac erythropoiesis, and scl-null embryonic stem cells do not contribute to hematopoiesis in chimeric mice. To analyze the molecular consequences of scl deficiency, we compared the gene expression profiles of control (wild-type and scl-heterozygous) and scl-null embryonic stem cells differentiated in vitro for up to 12 days. In control and scl-null embryoid bodies the temporal expression pattern of genes associated with the formation of ventral mesoderm, such as Brachyury, bone morphogenetic protein-4, and flk-1, was identical. Similarly, GATA-2, CD34, and c-kit, which are coexpressed in endothelial and hematopoietic lineages, were expressed normally in scl-null embryonic stem cell lines. However, hematopoietic-restricted genes, including the transcription factors GATA-1, EKLF, and PU.1 as well as globin genes and myeloperoxidase, were only expressed in wild-type and scl-heterozygous embryonic stem cells. Indirect immunofluorescence was used to confirm the observations that GATA-1 and globins were only present in control embryoid bodies but that CD34 was found on both control and scl-null embryoid bodies. These data extend the previous gene ablation studies and support a model whereby scl is absolutely required for commitment of a putative hemangioblast to the hematopoietic lineage but that it is dispensable for endothelial differentiation.

The effects of neuropeptides (calcitonin gene-related peptide and substance P) on cultured human pulp cells.

The sensory neuropeptides substance P and calcitonin gene-related peptide have been implicated in the mediation of pulpal inflammation. A possible role in healing following injury has also been suggested (Byers et al., 1990). This possibility has been investigated by an examination of a direct effect of substance P and calcitonin gene-related peptide in vitro on fibroblast-like cells derived from human dental pulp. Cells were cultured for 48 hr in Dulbecco's modified Eagle medium plus 20% fetal calf serum and antibiotics. Substance P and calcitonin gene-related peptide were added in the range from 10(-12) to 10(-4) mol/L. Fibroblast growth factor was used as a positive control. Effects on cell proliferation were assessed by cell counts (daily for 6 days) and [3H]-thymidine uptake (24 hr after the addition of peptides). An effect on cellular functional activity was measured by [35S]-sulfate incorporation into glycosaminoglycans, in confluent cell cultures. Both substance P and calcitonin gene-related peptide showed concentration-dependent stimulation of cell proliferation. The maximum stimulation of approximately 40% was achieved at substance P and calcitonin gene-related peptide concentrations of 10(-6) mol/L, comparable with stimulation by fibroblast growth factor. By contrast, little increase in glycosaminoglycan synthesis by confluent cells could be detected. The direct effect on pulp cells is consistent with a role of the neuropeptides in pulp healing. This is exerted at the level of cell proliferation, rather than functional activity.

Differential action of diffusible molecules in long-term marrow culture on proliferation of leukaemic and normal haemopoietic cells.

The capacity of diffusible molecules in the fluid phase of long-term human bone marrow culture (LTMC) to exert preferential adverse effects on leukaemic relative to normal haemopoietic cells has been investigated. Responses of isolated cell populations were assessed in diffusion chamber inserts which permitted contact with fluid phase molecules but not with the adherent stromal cell layer of the LTMC system. Growth of AML cells in diffusion chambers was inhibited during co-culture with LTMC of autologous leukaemic bone marrow, and the same effect was produced during co-culture with normal LTMC. No inhibitory action was exerted on growth of normal haemopoietic precursors under the same conditions. Comparable responses were observed with human leukaemic cell lines and patient leukaemic cells, and studies on cell lines demonstrated inhibition of growth was induced by molecules generated in LTMC which caused accumulation in G1 phase of leukaemic cells of both myeloid and lymphoid lineage. The inhibitory effect was not reproduced by TGF beta, IFN gamma, IFN alpha, TNF alpha, LIF, SCF or Il-6, and was not impaired by inhibitors of nitric oxide or PGE production in the LTMC. These observations suggest the action of diffusible molecules of undefined constitution contributes to the preferential loss of leukaemic cells in LTMC.

Contrasting patterns of neoplastic cell behaviour in long-term culture of bone marrow from patients with acute leukaemia and myelodysplastic disorders. A survey of responses in 31 cases with cytogenetic determination of neoplastic status of cultured cells in 17 studies.

Alterations in neoplastic cell behaviour responsible for increased production of terminally-differentiated granulocytes during long-term culture of bone marrow in different categories of acute leukaemia and myelodysplasia have been investigated. An increase in neutrophils associated with transition to a morphological picture identical to normal control cultures occurred in 15 of 25 studies on acute leukaemia in contrast to one of six studies on myelodysplastic disorders. An abnormal neoplastic karyotype was employed as a marker for monitoring the course of the neoplastic cell population in 11 studies in which there was progression towards a normal pattern of differentiation. An increase in differentiation was shown by this means to represent increased maturation of cells of the neoplastic process in one study on a myelodysplastic disorder, demonstrating domination of proliferative activity in culture by all of the myelodysplastic disorders examined. Transition towards normal differentiation in nine studies on acute leukaemia, however, correlated with partial or complete replacement of the acute leukaemic cells by normal haemopoietic series in de novo acute leukaemia, and by Ph positive cells in blast crisis of CML. Conversion to morphologically and cytogenetically normal cell populations in five studies on de novo acute leukaemia occurred in four cases which failed to respond to remission-induction therapy, suggesting the selective toxic effect capable of purging acute leukaemic cells from bone marrow operated by a mechanism which lacked cross-resistance to currently-employed cytotoxic agents.