Biochemical Journal

Acidic and basic fibroblast growth factors (aFGF and bFGF) are growth factors which may have a physiological role in the normal breast and in breast cancer. A study of the effects of aFGF and bFGF on a variety of breast cell lines and epithelial cells purified from normal breast organoids showed that whereas normal breast cells did not exhibit membrane ruffling in response to either of these growth factors, some breast cancer cell lines did. This difference was not due to lack of receptor since all the cell lines tested were mitogenically stimulated by bFGF. Dominant negative mutations of FGF receptor 3 (FGFR-3) and the small GTP-binding protein p21rac inhibited membrane ruffling, showing that receptor dimerization and phosphorylation and p21rac activation are prerequisites for membrane ruffling in response to aFGF and bFGF. Transient transfection of individual FGFRs into cos-7 cells showed that FGFR-1, FGFR-2 and FGFR-3 could not mediate a membrane ruffling response whereas FGFR-4 could. These studies elucidate one signalling mechanism of FGF and point to differences in the response of normal and cancer breast epithelial cells which may be important in cell motility.

The malaria parasite can synthesize haem de novo. In the present study, the expression of the parasite gene for Δ-aminolaevulinate synthase (PfALAS) has been studied by reverse transcriptase PCR analysis of the mRNA, protein expression using antibodies to the recombinant protein expressed in Escherichia coli and assay of ALAS enzyme activity in Plasmodium falciparum in culture. The gene is expressed through all stages of intra-erythrocytic parasite growth, with a small increase during the trophozoite stage. Antibodies to the erythrocyte ALAS do not cross-react with the parasite enzyme and vice versa. The recombinant enzyme activity is inhibited by ethanolamine and the latter inhibits haem synthesis in P. falciparum and growth in culture. The parasite ALAS is localized in the mitochondrion and its import into mitochondria in a cell-free import assay has been demonstrated. The import is blocked by haemin. On the basis of these results, the following conclusions are arrived at: PfALAS has distinct immunological identity and inhibitor specificity and is therefore a drug target. The malaria parasite synthesizes haem through the mitochondrion/cytosol partnership, and this assumes significance in light of the presence of apicoplasts in the parasite that may be capable of independent haem synthesis. The PfALAS gene is functional and vital for parasite haem synthesis and parasite survival.

When Fe2+ ions are added to rat-liver microsomes, lipid peroxidation begins after a short lag period. Fe2+-dependent peroxidation in the first few minutes of the incubation can be increased by adding Fe3+, ascorbic acid or Pb2+ ions; these stimulations are not additive. By contrast, Pb2+ ions inhibit peroxidation of microsomes in the presence of Fe3+/ascorbate or Fe3+-ADP/NADPH. In liposomes made from ox-brain phospholipids, Fe2+-dependent peroxidation is stimulated slightly by Fe3+, but much more so by ascorbic acid, Al3+ or Pb2+; these stimulations are not additive. Liposomal peroxidation in the presence of Fe3+/ascorbate is inhibited by Pb2+ or Al3+. These results argue against the participation of an Fe2+-Fe3+-O2 complex, or a critical 1:1 ratio of Fe2+ to Fe3+, in the initiation of lipid peroxidation in liposomes and rat-liver microsomes.

MAC (mitochondrial apoptosis-induced channel) forms in the mitochondrial outer membrane and unleashes cytochrome c to orchestrate the execution of the cell. MAC opening is the commitment step of intrinsic apoptosis. Hence closure of MAC may prevent apoptosis. Compounds that blocked the release of fluorescein from liposomes by recombinant Bax were tested for their ability to directly close MAC and suppress apoptosis in FL5.12 cells. Low doses of these compounds (IC50 values ranged from 19 to 966 nM) irreversibly closed MAC. These compounds also blocked cytochrome c release and halted the onset of apoptotic markers normally induced by IL-3 (interleukin-3) deprivation or staurosporine. Our results reveal the tight link among MAC activity, cytochrome c release and apoptotic death, and indicate this mitochondrial channel is a promising therapeutic target.

Increases in mitochondrial [Ca2+] ([Ca2+]m) have recently been reported to cause long-term alterations in cellular ATP production [Jouaville, Bastianutto, Rutter and Rizzuto (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 13807Ő13812]. We have determined the importance of this phenomenon for nutrient sensing in pancreatic islets and β-cells by imaging adenovirally expressed Ca2+ and ATP sensors (aequorin and firefly luciferase). [Ca2+]m increases provoked by KCl or tolbutamide evoked an immediate increase in cytosolic and mitochondrial free ATP concentration ([ATP]c and [ATP]m respectively) at 3mM glucose. Subsequent increases in [glucose] (to 16 or 30mM) then caused a substantially larger increase in [ATP]c and [ATP]m than in naïve cells, and pre-stimulation with tolbutamide led to a larger secretory response in response to glucose. Whereas pre-challenge of islets with KCl altered the response to high [glucose] of [Ca2+]m from periodic oscillations to a sustained elevation, oscillations in [ATP]c were observed neither in naïve nor in stimulated islets. Hence, long-term potentiation of mitochondrial ATP synthesis is a central element in nutrient recognition by pancreatic islets.

The human hepatoma cell line Hep G2 was studied with respect to metabolism of human low-density lipoprotein (LDL). The Hep G2 cells bind, take up and degrade human LDL with a high-affinity saturable and with a low-affinity non-saturable component. The high-affinity binding possesses a KD of 25 nM-LDL and a maximal amount of binding of about 70 ng of LDL-apoprotein/mg of cell protein. The high-affinity binding, uptake and degradation of LDL by Hep G2 cells is dependent on the extracellular Ca2+ concentration and is down-regulated by the presence of fairly high concentrations of extracellular LDL. Incubation of the Hep G2 cells with LDL results in suppression of the intracellular cholesterol synthesis. It is concluded that the human hepatoma cell line Hep G2 possesses specific LDL receptors similar to the LDL receptors demonstrated on extrahepatic tissue cells.

COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3´ untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a β-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant sequences were identified that were incapable of destabilizing a reporter mRNA, yet showed unimpaired binding of FBP1 and AUF-1 and/or -2. TTP was absent from the HeLa cell line used in this analysis. Finally, RNA interference experiments argued against a prominent role for HuR in the CR1-mediated regulation of mRNA stability. We conclude that at least one critical regulator of COX-2 mRNA stability is likely to remain unidentified at present.

This study describes the identification of Drosophila yellow-f and yellow-f2 as dopachrome-conversion enzymes responsible for catalysing the conversion of dopachrome into 5,6-dihydroxyindole in the melanization pathway. Drosophilayellow-y gene and yellow-b, -c, -f and -f2 genes were expressed in an insect cell/baculovirus expression system and their corresponding recombinant proteins were screened for dopachrome-conversion enzyme activity. Among the yellow and yellow-related genes, the yellow-f and yellow-f2 genes were identified as the genes coding for Drosophila dopachrome-conversion enzyme based on the high activity of their recombinant proteins in catalysing the production of 5,6-dihydroxyindole from dopachrome. Both yellow-f and yellow-f2 are capable of mediating a decarboxylative structural rearrangement of dopachrome, as well as an isomerization/tautomerization of dopamine chrome and dopa methyl ester chrome. Northern hybridization revealed the transcription of yellow-f in larvae and pupae, but a high abundance of mRNA was observed in later larval and early pupal stages. In contrast, yellow-f2 transcripts were present at all stages, but high abundance of its mRNA was observed in later-stage pupae and adults. These data indicate that yellow-f and yellow-f2 complement each other during Drosophila development and that the yellow-f is involved in larval and pupal melanization, and yellow-f2 plays a major role in melanization reactions in Drosophila during later pupal and adult development. Results from this study provide the groundwork towards a better understanding of the physiological roles of the Drosophilayellow gene family.

A simple method for preparing plasma membranes from bovine testes is described. Bovine testicular receptor has a high affinity and specificity for 125I-labelled human FSH (follicle-stimulating hormone). The specific binding of 125I-labelled human FSH to the plasma membranes is a saturable process with respect to the amounts of receptor protein and FSH added. The association and dissociation of 125I-labelled human FSH are time- and temperature-dependent, and the binding of labelled human FSH to bovine testicular receptor is strong and not readily reversible. Scatchard [Ann. N.Y. Acad. Sci. (1949) 51, 660-672] analysis indicates a dissociation constant, Kd, of 9.8 ×10-11M, and 5.9 × 10-14mol of binding sites/mg of membrane protein. The testicular membrane receptor is heat-labile. Preheating at 40°C for 15 min destroyed 30% of the binding activity. Specific binding is pH-dependent, with an optimum between pH 7.0 and 7.5. Brief exposure to extremes of pH caused irreversible damage to the receptors. The ionic strength of the incubation medium markedly affects the association of 125I-labelled human FSH with its testicular receptor. Various cations at concentrations of 0.1M inhibit almost completely the binding of 125I-labelled human FSH. Nuclectides and steroid hormones at concentrations of 1mM and 5 μg/ml respectively have no effect on the binding of FSH to its receptor. Incubation of membranes with and chymotrypsin resulted in an almost complete loss of binding activity, suggesting that protein moieties are essential for the binding of 125I-labelled human FSH. Binding of 125I-labelled human FSH to bovine testicular receptor does not result in destruction or degradation of the hormone.

Two members of the NF-κB (nuclear factor κB)/Rel transcription factor family, NF-κB1 and NF-κB2, are produced as precursor proteins, NF-κB1 p105 and NF-κB2 p100 respectively. These are proteolytically processed by the proteasome to produce the mature transcription factors NF-κB1 p50 and NF-κB2 p52. p105 and p100 are known to function additionally as IκBs (inhibitors of NF-κB), which retain associated NF-κB subunits in the cytoplasm of unstimulated cells. The present review focuses on the latest advances in research on the function of NF-κB1 and NF-κB2 in immune cells. NF-κB2 p100 processing has recently been shown to be stimulated by a subset of NF-κB inducers, including lymphotoxin-β, B-cell activating factor and CD40 ligand, via a novel signalling pathway. This promotes the nuclear translocation of p52-containing NF-κB dimers, which regulate peripheral lymphoid organogenesis and B-lymphocyte differentiation. Increased p100 processing also contributes to the malignant phenotype of certain T- and B-cell lymphomas. NF-κB1 has a distinct function from NF-κB2, and is important in controlling lymphocyte and macrophage function in immune and inflammatory responses. In contrast with p100, p105 is constitutively processed to p50. However, after stimulation with agonists, such as tumour necrosis factor-α and lipopolysaccharide, p105 is completely degraded by the proteasome. This releases associated p50, which translocates into the nucleus to modulate target gene expression. p105 degradation also liberates the p105-associated MAP kinase (mitogen-activated protein kinase) kinase kinase TPL-2 (tumour progression locus-2), which can then activate the ERK (extracellular-signal-regulated kinase)/MAP kinase cascade. Thus, in addition to its role in NF-κB activation, p105 functions as a regulator of MAP kinase signalling.