Abstract The release of various fatty acids (FAs) from permeabilized HL-60 cells, predominantly oleic acid (OA) rather than arachidonic acid, was greatly enhanced by GTP-γ-S and vanadate [Tsujishita, Y., Asaoka, Y. and Nishizuka, Y., Proc. Natl. Acad. Sci. USA 91 (1994) 6274–6278]. The present study shows that phospholipase A (A2/A1) activity which cleaves the acyl group from both sn-2 and sn-1 positions of phosphatidylethanolamine (PtdEtn) is increased in HL-60 cells during differentiation to granulocyte-like cells. This enzyme does not require Ca2+ and releases various FAs, preferentially OA from PtdEtn and, to lesser extent, from lysoPtdEtn. Other phospholipids including phosphatidylcholine and phosphatidic acid serve as very poor substrates. Although further studies are necessary to show the direct link of this enzyme activation to receptor stimulation, the results described here imply that this enzyme is responsible for the release of various FAs, particularly OA, from permeabilized HL-60 cells.

Abstract We have recently shown the presence of αB-crystallin in non-ocular tissues of diverse embryological origins such as the heart, brain, spinal cord, kidney, retina, etc. Using an αB-crystallin-specific antiserum and immunofluorescence, immunoblotting, immunoprecipitation and peptide mapping with Staphylococcus aureus protease, we demonstrate differential expression of αB-crystallin in epithelial and fibroblast cell lines. αB-Crystallin was detectable only in epithelial cell lines such as MDBK, MDCK, LLCPK1 and JTC-12, and was not observed in two kidney fibroblast cell lines, one skin fibroblast cell line, and one corneal fibroblast cell line. Differential expression of the αB-crystallin gene was also confirmed by Northern blot analysis of the RNAs isolated from these cell lines. These data suggest a cell-type-specific role for αB.

Abstract Proneural genes control the generation of neuroblasts from the neuroepithelium, but their functions in neuroblast asymmetric division and migration remain elusive. Here, we identified Caenorhabditiselegans mutants of a proneural transcription factor (TF) lin-32, in which Q neuroblasts are produced. We showed that LIN-32 functions in parallel with a storkhead TF, HAM-1, to regulate Q neuroblast asymmetric division, and that Q neuroblast migration is inhibited in lin-32 alleles. Consistently, lin-32 is expressed throughout Q neuroblast lineage, suggesting that LIN-32 may promote different target gene expression. Our studies thus uncovered previously unknown functions of a proneural gene in neuroblast development.

Abstract We report reversible Ca2+-induced Ca2+ release from mitochondria, which takes the form of Ca2+ spikes. Mitochondrial Ca2+ spiking is an all-or-none process with a threshold dependence on both the frequency and the amplitude of the Ca2+ pulses used as stimuli. This spiking relies on the transient operation of the mitochondrial permeability transition pore, and is initiated — in a threshold-dependent manner — with inositol-triphosphate-mediated Ca2+ responses in permeabilized cells. Evidence that mitochondrial Ca2+-induced Ca2+ release contributes to inositol-triphosphate-mediated Ca2+ responses in intact cells is also reported.

Abstract Chick periosteum-derived cells, which do not enter the chondrogenic cell lineage during normal bone development and growth, exhibit chondrogenic potential in high cell density culture conditions. In such cultures, collagen gene expression was temporally analyzed at the mRNA level by a reverse transcription PCR (RT-PCR) procedure, which showed that α1(II) and α1(IX) collagen mRNAs are coordinately increased, coincident with the onset of overt chondrogenesis, and subsequently decreased as chondrocytes exhibited hypertrophic characteristics. α1(X) collagen mRNA was detected well before the onset of chondrogenesis and markedly increased along with the hypertrophic change. For α2(I) collagen, both the bone/tendon form and the cartilage form of mRNA were detected throughout the culture period. This culture system provides an experimental vehicle capable of investigating the molecular events involved in the full range of chondrogenic differentiation starting from uncommitted periosteum-derived mesenchymal stem cells.

Abstract The observed homology between G-proteins which regulate adenylate cyclase and ras proteins and the suggested role of ras in the regulation of adenylate cyclase in yeast prompted us to examine the regulation of adenylate cyclase in three cell lines: (i) NIH 3T3 cells, (ii) NIH 3T3 cells transformed by high levels of the normal rasH gene product and (iii) NIH 3T3 cells transformed by a mutated rasH gene product. We found that the regulation of adenylate cyclase by G-proteins is identical in the three cell lines, although the response of the transformed NIH 3T3 cells to agonists is strongly attenuated. Our data suggest that mammalian ras products do not interact directly with adenylate cyclase, although their increased expression may indirectly inhibit the interaction of adenylate cyclase stimulatory receptors with G-proteins.

Abstract Muscle glycogen synthesis is modulated by physiologically relevant changes in cell volume. We have investigated the possible involvement of integrin-extracellular matrix interactions in this process using primary cultures of rat skeletal muscle subject to hypo- or hyper-osmotic exposure with integrin binding peptide GRGDTP to disrupt integrin actions and the inactive analogue GRGESP as control. Osmotically induced increases (77%) and decreases (34%) in glycogen synthesis (d-[14C]-glucose incorporation into glycogen) were prevented by GRGDTP (but not GRGESP) without affecting glucose transport. Cytoskeletal disruption with cytochalasin D or colchicine had similar effects to GRGDTP. Osmotically induced modulation of muscle glycogen synthesis involves integrin-extracellular matrix interactions and cytoskeletal elements, possibly as components of a cell-volume `sensing' mechanism.

Abstract In a proteomics-based screen for proteins interacting with cyclin-dependent protein kinase (CDK), we have identified a novel CDK complex containing the eukaryotic translation initiation factor, eIF4A. Reciprocal immunoprecipitations using antibodies against eIF4A indicate that the interaction is specific. The CDKA–eIF4A complex is abundant in actively proliferating and growing cells but is absent from cells that have ceased dividing. The CDKA–eIF4A complex contains kinase activity that is sensitive to the CDK-specific inhibitor roscovitine. This interaction points to a possible molecular mechanism linking cell proliferation with translational control.

Abstract During the acute phase response, cytokines induce hepatic metallothionein and ceruloplasmin synthesis and the uptake of metals. We have investigated how copper and cytokines may interact in controlling ceruloplasmin (CP) and metallothionein mRNA in liver cells. We found that IL-1α, IL-1β and IL-6 increased both metallothionein-1 (MT-1) and metallothionein-2 (MT-2) mRNA in HepG2 cells. The time and pattern of induction was different, both IL-1α and IL-1β inducing two peaks of MT-1 and MT-2, with that of MT-2 being much larger. IL-6 induced only low levels of both MT-1 and MT-2 mRNA. CP mRNA was also increased after 16 h by IL-1β, whereas IL-1α induced two CP peaks at 8 and 20 h, while IL-6 had little effect. Copper administration gave rise to substantially increased MT-1 mRNA, a slightly lower increase in MT-2 and also a significant increase in CP mRNA with similar kinetics. These parallel increases in MT and CP mRNA suggest that the coordinated expression of these proteins may be important for their synthesis during the acute phase response.

Abstract We investigated the existence and possible role of cytosolic phospholipase A2 (cPLA2) in rat decidualized uteri. PLA2 activity in the cytosol of a decidualized uterine horn, induced by intraluminal oil infusion, was significantly higher than that in contralateral intact horn. The activity was almost completely depressed by cPLA2 inhibitors including arachidonyl trifluoromethyl ketone (ATK). The immunoreactive signals for cPLA2 were intense in decidua and glandular epithelial cells. In vivo administration of ATK (0.1–100 μg) caused a dose-dependent inhibition of decidualization. These results show the presence of cPLA2 and its probable implication in decidualization in rat uterus.