Effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of osteoblastic clone MC3T3-e1 cells.

The effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of a clonal osteoblastic cell line, MC3T3-E1, were investigated. The toxin induced a morphological change in the cells from a spindle shape to a spherical form with many blebs. The toxin-treated cells were viable and grew to form confluent cell layers composed of irregularly shaped cells and multinuclear cells. The toxin inhibited elevation of alkaline phosphatase activity in the cells in a dose-dependent manner at concentrations from 10 pg to 10 ng/ml. The accumulation of type I collagen in the cells was also reduced by the toxin. Since high alkaline phosphatase activity and accumulation of collagen are closely linked to differentiation of the cells into osteoblasts, it is considered likely that B. bronchiseptica dermonecrotic toxin impairs the ability of the cells to differentiate.     

Correlation between ribosylation of pertussis toxin substrates and inhibition of peptidoglycan-, muramyl dipeptide- and lipopolysaccharide-induced mitogenic stimulation in B lymphocytes

Selective inhibition by pertussis toxin (PT) of mitogenic activation of mouse B lymphocytes by bacterial mitogens (peptidoglycan and lipopolysaccharide) and muramyl dipeptide (a synthetic analog of peptidoglycan fragment) was demonstrated. Mitogenic activation of B cells by protein kinase C activators and ionomycin was insensitive to PT. Also PT did not inhibit peptidoglycan- and lipopolysaccharide-induced differentiation of B cells into Ig-secreting cells, when it was added to the cultures after the proliferative stage of the response. B lymphocyte membranes contained two major PT substrates (40 and 41 kDa). The extent of PT-mediated ADP ribosylation of these substrates correlated with the degree of PT-mediated inhibition of mitogenic stimulation of B cells. B cell stimulation by all mitogens tested was not inhibited by cholera toxin at nontoxic concentrations that are known to cause maximal increase in cAMP in B cells. Since the only known substrates for PT-mediated ADP ribosylation in mammalian cells are the alpha subunits of some G proteins, our data suggest that G proteins are present in B cell membranes and that they are involved in B cell activation induced by bacterial mitogens.     

Expression of parathyroid hormone-related peptide (PthrP) and its receptor (PTH1R) during the histogenesis of cartilage and bone in the chicken mandibular process

The purpose of this study was to examine the expression and actions of parathyroid hormone-related protein (PTHrP) when skeletal histogenesis occurs in the chicken mandible. Prior to the appearance of skeletal tissues, PTHrP and PTH1R were co-expressed by cells in the ectoderm, skeletal muscle, peripheral nerve and mesenchyme. Hyaline cartilage was first observed at HH stage 27 when many but not all chondroblasts expressed PTHrP and PTH1R. By stage 34, PTHrP and PTH1R were not detected in chondrocytes but were expressed in the perichondrium. Alkaline phosphatase (AP)-positive preosteoblasts and woven bone appeared at stages 31 and 34, respectively. Preosteoblasts, osteoblasts and osteocytes co-expressed PTHrP and PTH1R. Treatment with chicken PTHrP (1-36) increased cAMP in mesenchyme from stage 26 embryos. Continuous exposure to chicken PTHrP (1-36) for 14 days increased cartilage nodule number and decreased AP while intermittent exposure did not affect cartilage nodule number and increased AP in cultures of stage 26 mesenchymal cells. Adding a neutralizing anti-PTHrP antibody to the cultures reduced cartilage nodule number and did not affect AP. These findings show that PTHrP and PTH1R are co-expressed by extraskeletal and skeletal cells before and during skeletal tissue histogenesis, and that PTHrP may influence skeletal tissue histogenesis by affecting the differentiation of mandibular mesenchymal cells into chondroblasts and osteoblasts.     

Effect of Pasteurella multocida toxin on bone resorption in vitro.

Pasteurella multocida toxin (PMT), which is the primary etiologic factor in the pathogenesis of progressive atrophic rhinitis in pigs, was found to stimulate bone resorption in vitro. This stimulation was observed both in cultures of murine calvaria by measuring the release of calcium and of the lysosomal enzyme beta-glucuronidase and in murine long bone cultures by measuring the release of calcium. Both systems showed the same dose response curve, with the maximal effect at a concentration of 5 ng/ml. The effect on calvaria was studied in more detail. PMT increased bone resorption 24 h after its addition and always had to be present to express an effect. Calcitonin was able to inhibit this increase of resorption completely, and inhibitors of prostaglandin synthesis suppressed it partially. Although the data show an effect of PMT on bone tissue, the results do not exclude an action on cells in the nasal cavity, which could indirectly stimulate bone resorption.     

Stimulatory and inhibitory effects of cyclic AMP on lymphocytes from atopic children.

The effect of cholera toxin and dibutyryl cAMP on mitogen-activated lymphocytes from atopic and non-atopic individuals was studied. Cholera toxin enhanced stimulation by phytohemagglutinin of cells from small children but not from adults. Dibutyryl cAMP at low concentration (less than 10(-5) M) significantly enhanced the lymphocyte response to mitogens in some, but not all individuals. High concentrations, on the other hand, were consistently inhibitory. In atopic children, the lymphocyte response to T cell mitogen was significantly less stimulated by cholera toxin, and more inhibited by dibutyryl cAMP than the response of cells from non-atopic matched controls. Thus, T cells from atopic individuals appear to have an altered sensitivity to the action of cAMP, possibly resulting in an impaired balance between helper and suppressor T cells. The hypothesis is advanced, that such an altered balance is causally related to hyperproduction of IgE resulting in atopic disease.     

Endorphin suppresses FSH-stimulated proliferation of isolated neonatal Sertoli cells by a pertussis toxin-sensitive mechanism

During perinatal development, when the size of the Sertoli cell population is determined, Leydig cells produce beta-endorphin, a peptide which may interact with Sertoli cells to modify their FSH-responsiveness, as suggested by our previous work. The goal of the present study was first, to test directly the possibility that beta-endorphin modifies the proliferative response of neonatal Sertoli cells to FSH, and second, to gain information on a mechanism(s) involved in any observed effect. We treated isolated 6-day-old Sertoli cells with FSH or vehicle in vitro and measured their incorporation of exogenous, radiolabeled thymidine with quantitative autoradiography. After 2 days in culture with FSH, we detected a 10-fold increase in the rate of Sertoli cell proliferation. The level of cell division in these FSH-treated cultures was identical to that in other cultures exposed to cAMP under similar conditions. In addition, inclusion of beta-endorphin 3 hr prior to FSH or cAMP decreased the effect of the hormone by 50% but left the cAMP response unchanged. Thus, beta-endorphin acts on isolated, neonatal Sertoli cells at a point prior to intracellular production of cAMP to suppress their response to FSH. When other cultures were treated with pertussis toxin, a blocker of intracellular GTP-binding proteins such as Gi, before sequential addition of endorphin and FSH, the effect of beta-endorphin on FSH-responsiveness was abolished. Moreover, when other cultures were exposed to pertussis toxin in the absence of endorphin, followed by FSH, their response to the hormone was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pasteurella multocida Toxin as a Transporter of Non-Cell-Permeating Proteins

The protein toxin Pasteurella multocida toxin (PMT) is the causative agent of atrophic rhinitis in pigs, leading to atrophy of the nasal turbinate bones by affecting osteoblasts and osteoclasts. The mechanism of PMT-induced intoxication is a deamidation of α-subunits of heterotrimeric G proteins, including Gα_q, Gα₁₃, and Gα_i, thereby causing persistent activation of the G proteins. Here we utilized PMT as a transporter of the non-cell-permeating A domain of diphtheria toxin (DTa). Fusion proteins of PMT and DTa ADP-ribosylated elongation factor 2, the natural target of diphtheria toxin, leading to cell toxicity. PMT-DTa effects were competed by PMT, indicating binding to the same cell surface receptor. Fluorescently labeled PMT-DTa and PMT colocalized with specific markers of early and late endosomes. Bafilomycin A, which inhibits vacuolar H⁺-ATPase, blocked PMT-DTa-induced intoxication of HEK-293 cells. By constructing various PMT-DTa chimeras, we identified a minimal region of PMT necessary for uptake of DTa. The data suggest that PMT is able to transport cargo proteins into eukaryotic cells by utilizing the PMT-specific uptake route.     

Inhibition of secretion of staphylococcal alpha toxin by cerulenin

Secretion of alpha toxin by Staphylococcus aureus strain Wood 46 was preferentially inhibited by cerulenin, an antibiotic that stops fatty-acid synthesis by inhibiting beta-keto acyl acyl carrier-protein synthetase. At the concentrations used, cerulenin had a negligible effect on cell growth and total protein synthesis, but reduced lipid synthesis by 50%. Extracellular and membrane-associated alpha toxin was absent in cultures treated with cerulenin, but toxin formation was resumed after either removal of the antibiotic or addition of exogenous fatty acids. The apparent absence of toxin precursor in membranes of inhibited cells favours inhibition at an earlier stage in toxin synthesis.     

Anthrax edema toxin requires influx of calcium for inducing cyclic AMP toxicity in target cells

The anthrax edema toxin comprises two proteins: protective antigen and edema factor. Anthrax protective antigen binds to the receptors on the surface of target cells and facilitates the entry of edema factor into these target cells. Edema factor (EF) is an adenylate cyclase that catalyzes the synthesis of cyclic AMP (cAMP) in the cytosol of the host cells. In this study, we examined the requirement of extracellular calcium for anthrax edema toxin-induced toxicity in host cells. The cAMP response generated by edema toxin was analyzed in a variety of cells, including CHO, macrophage-like RAW264.7, human neutrophils, and human lymphocytes. Our investigations reveal that after EF reaches the cell cytosol, a rapid influx of calcium is triggered in the host cell that has a pivotal role in determining the cAMP response of the affected cells. Although the cAMP response generated by edema toxin in different cell types varied in intensity and in the time of initiation, the influx of calcium invariably preceded cAMP accumulation. Agents that blocked the uptake of calcium also inhibited edema toxin-induced accumulation of cAMP in the host cells. This is the first report that demonstrates that edema toxin induces accumulation of cAMP in lymphocytes. By accumulating cAMP, a potent inhibitor of immune cell function, edema toxin may actually be poisoning the immune system and thus facilitating the survival of the bacteria in the host.     

Cyclic AMP inhibits increased collagen production by cyclically stretched smooth muscle cells

Rabbit arterial smooth muscle cells, grown on elastin membranes which were cyclically elongated and relaxed, responded by increasing their rates of synthesis of protein and, in particular, of collagen, compared to stationary controls. Raising intracellular cyclic AMP (cAMP) levels by adding theophylline or dibutyryl cAMP to the culture medium prevented the synthetic response to cyclic stretching, but did not alter the rates of protein or collagen synthesis by stationary controls. Both synthesis and degradation of collagen by cyclically stretched cells increased in parallel such that the proportion of synthesized collagen that was degraded was similar to that found in the stationary cultures. Collagen degradation was not affected by theophylline administration to stationary cell cultures but the drug increased degradation of collagen by cyclically stretched cells. We conclude that the net production of protein, and in particular of a structural protein, collagen, by arterial smooth muscle cells subjected to the mechanical force of stretching was inhibited when intracellular levels of cAMP were raised. The results suggest that cAMP may play a role in the modulation of structural protein content of artery walls in response to changes in tensile stress.     

Biological activity of a C-terminal fragment of Pasteurella multocida toxin

The protein toxin of Pasteurella multocida PMT is a potent mitogen and activator of phospholipase Cbeta. In this study different toxin fragments were investigated. A C-terminal fragment encompassing amino acids 581 through 1285 (PMT581C) was constructed, which was inactive toward intact embryonic bovine lung (EBL) cells after addition to culture medium but caused reorganization of the actin cytoskeleton and rounding up of cells when introduced into the cells by electroporation. As the holotoxin, the toxin fragment PMT581C induced an increase in total inositol phosphate levels after introduction into the cell by electroporation. A C-terminal fragment shorter than PMT581C as well as N-terminal fragments were inactive. Exchange of cysteine-1165 for serine in the holotoxin resulted in a complete loss of the ability to increase inositol phosphate levels. Correspondingly, the mutated toxin fragment PMT581C.C1165S was inactive after cell introduction by electroporation, suggesting an essential role of Cys-1165 in the biological activity of the toxin.     

Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

The locations of the catalytic and receptor-binding domains of the Pasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed alpha/beta domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.     

Tumor necrosis factor-alpha is chemokinetic for lymphokine-activated killer cells: regulation by cyclic adenosine monophosphate.

The ability of tumor necrosis factor-alpha (TNF-alpha) to attract lymphokine-activated killer (LAK) cells in vitro was examined. Utilizing modified Boyden chambers (BC), it was observed that TNF-alpha is not chemoattractant for LAK cells. On the other hand, TNF-alpha attracted both fresh and concanavalin A-activated T cells. However, when TNF-alpha was incubated in the upper compartments of BC and in the presence of LAK cells, it enhanced the random movement of these cells across the polycarbonate membranes. The effect of TNF-alpha was inhibited by incorporating anti-TNF-alpha antibody, or a high concentration (10 ng) of TFG-beta 1. The activity of TGF-beta 1 was reversed by anti-TGF-beta 1 antibody. Cholera toxin (CT), which is known to activate the endogenous level of cyclic adenosine monophosphate (cAMP) also inhibited TNF-alpha-induced LAK cell chemokinesis. The effect of CT was mimicked by the cAMP analog dibutyryl cAMP or by the phosphodiesterase inhibitors isobutyl methylxanthine or aminophylline. Measurement of the intracellular level of cAMP showed that cells incubated for 1, 2, or 4 hr with TNF-alpha have a lower level of cAMP, whereas those incubated with a high concentration of TGF-beta 1 produced significantly higher levels of this messenger. cAMP level was also increased in cells incubated with TGF-beta 1 plus TNF-alpha. This level was reduced to the background when anti-TGF-beta 1 antibody was added to the cultures. These results suggest that cAMP negatively regulates LAK cell chemokinesis.     

PD98059对人骨髓间质干细胞分化为成骨细胞的影响

目的:探讨胞外信号调节激酶(ERK)信号传导途径对骨髓间质干细胞(MSC)分化为成骨细胞的影响。方法:采用Ficoll-Paque淋巴细胞分离液分离成人MSC,体外扩增,应用地塞米松、β-甘油磷酸钠、vitaminC定向诱导MSC分化为成骨细胞。在成骨诱导液中加入不同剂量的PD98059,观察其对成骨细胞形成的影响。结果:MSC体外扩增15代可获得(3-4)×10¹²个细胞。在成骨诱导液作用下,MSC可在体外定向分化为成骨细胞。不同剂量的PD98059均可抑制MSC分化为成骨细胞,并有剂量依赖关系;同时促使部分细胞转化为脂肪细胞。结论:ERK信号传导途径可能在MSC分化为成骨细胞和脂肪细胞过程中起关键作用。     

The pasteurella multocida toxin interacts with signalling pathways to perturb cell growth and differentiation

Some years ago we showed that the Pasteurella multocida toxin (PMT) is a potent mitogen for cells in culture. It is an intracellularly acting toxin that stimulates several signal transduction pathways. The heterotrimeric G-protein, Gq, is stimulated, which in turn causes activation of protein kinase C and an increase in inositol trisphosphates. The Rho GTPase is also activated, leading via the Rho kinase, to activation of the focal adhesion kinase and to cytoskeletal rearrangements. Analysis of the PMT sequence suggested the presence of three domains that encode receptor binding, translocation and catalytic domains. The location of all three domains has been confirmed directly. Competitive binding assays confirmed that the N-terminus of PMT encoded the receptor-binding domain, while cytoplasmic microinjection of expressed PMT fragments identified the location of the C-terminal catalytic domain. Recently, we have demonstrated the presence of key amino acids that affect membrane insertion within the putative transmembrane domain. Several lines of evidence suggest that PMT activates Galphaq, and that this is one potential molecular target for the toxin. Galphaq is known to be tyrosine phosphorylated when activated normally via a G-protein-coupled receptor (GPCR), and it has been suggested that this is an essential part of the activation process. We have shown that PMT induces Galphaq tyrosine phosphorylation, but that this is not essential for activation of the G-protein. Furthermore, a totally inactive mutant of PMT stimulates Galpha phosphorylation without leading to its activation. Phosphorylation of Galphaq triggered by the inactive mutant potentiates activation of Gq via a GPCR, demonstrating that phosphorylation of Gq cannot lead to receptor uncoupling. Natural or experimental infection of animals with toxigenic P. multocida, or injection with purified recombinant PMT causes loss of nasal turbinate bone. The effects on bone have been analysed in vitro using cultures of osteoblasts--cells that lay down bone. PMT blocks the formation of mature calcified bone nodules and the expression of differentiation markers such as CBFA-1, alkaline phosphatase and osteocalcin. These effects can be partially prevented by inhibitors of Rho or Rho kinase function, implicating this pathway in osteoblast differentiation. Indeed, inhibitors of Rho stimulate the formation of bone nodules in vitro. In summary, PMT is a novel toxin that acts via signalling pathways to promote proliferation in many cells, while specifically inhibiting differentiation in osteoblast cells.     

Different effect of prostaglandin E2 on B-cell activation by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2: selective inhibition of B151-TRF2-induced antibody response through increases in intracellular cyclic AMP levels

Effects of prostaglandin E2 (PGE2) on murine B-cell activation induced by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2, were examined. A final differentiation of unprimed B cells into IgM-producing cells induced by B151-TRF2 was markedly inhibited by PGE2 at physiological concentrations (around 10(-8) M), whereas B151-TRF1/IL-5- induced antibody responses of unprimed as well as activated B cells were not affected by PGE2, even at 10(-6) M. B-cell responses induced by B151-TRF2-like factors from autoimmune-prone MRL/lpr mice were also inhibited by PGE2. Biphasic increases in intracellular cyclic AMP (cAMP) levels were induced by culturing B cells with 10(-6) or 10(-8) M PGE2: rapid increases within 8 min and delayed increases around 16 hr. The direct addition of dibutyryl cAMP to cultures of B cells resulted in marked inhibition of antibody responses when stimulated with B151-TRF2 but not with B151-TRF1/IL-5. The B151-TRF2-induced antibody responses were also inhibited by cAMP-elevating reagents such as forskolin, cholera toxin and theophylline. Furthermore, 2'.5'-dideoxyadenosine, which is an inhibitor of adenylate cyclase, prevented the PGE2-mediated cAMP accumulation in unprimed B cells as well as the PGE2-mediated inhibition of B151-TRF2-induced B-cell responses when added at the initiation of culture. These results suggest that PGE2 inhibits B151-TRF2-induced antibody responses through the activation of adenylate cyclase and subsequent accumulation of intracellular cAMP, whereas B151-TRF1/IL-5-responsive B cells are resistant to the inhibitory effect of PGE2 and cAMP.     

Phagocytosis of wear debris by osteoblasts affects differentiation and local factor production in a manner dependent on particle composition

Wear debris is considered to be one of the main factors responsible for aseptic loosening of orthopaedic endoprostheses. Whereas the response of cells in the monocytic lineage to foreign materials has been extensively studied, little is known about cells at the bone formation site. In the present study, we examined the hypothesis that the response of osteoblasts to wear debris depends on the chemical composition of the particles. We produced particles from commercially pure titanium (cpTi), Ti-6Al-4V (Ti-A), and cobalt-chrome (CoCr) and obtained ultrahigh molecular weight polyethylene (UHMWPE; GUR 4150) particles from a commercial source. The equivalent circle diameters of the particles were comparable: 1.0 +/- 0.96 microm for UHMWPE; 0.84 +/- 0.12 microm for cpTi; 1.35 +/- 0.09 microm for Ti-A, and 1.21 +/- 0.16 microm for CoCr. Confluent primary human osteoblasts and MG63 osteoblast-like cells were incubated in the presence of particles for 24 h. Harvested cultures were examined by transmission electron microscopy to determine if the cells had phagocytosed the particles. Particles were found intracellularly, primarily in the cytosol, in both the primary osteoblasts and MG63 cells. The chemical composition of the particles inside the cells was confirmed by energy-dispersive X-ray analysis. Morphologically, both cell types had extensive ruffled cell membranes, less-developed endoplasmic reticulum, swollen mitochondria, and vacuolic inclusions compared with untreated cells. CpTi, Ti-A, and CoCr particles were also added to cultures of MG63 cells to assess their effect on proliferation (cell number) and differentiation (alkaline phosphatase activity), and PGE2 production. All three types of particles had effects on the cells. The effect on cell number was dependent on the chemical composition of the particles; Ti-A and CoCr caused a dose-dependent increase, while cpTi particles had a biphasic effect with a maximal increase in cell number observed at the 1:10 dilution. Alkaline phosphatase specific activity was also affected and cpTi was more inhibitory than Ti-A or CoCr. PGE2 production was increased by all particles, but the magnitude of the effect was particle-dependent: CoCr > cpTi > Ti-A. This study demonstrates clearly that human osteoblast-like cells and MG63 cells can phagocytose small UHMWPE, CoCr, Ti-A, and cpTi particles. Phagocytosis of the particles is correlated with changes in morphology, and analysis of MG63 response shows that cell proliferation, differentiation, and prostanoid production are affected. This may have negative effects on bone formation adjacent to an orthopaedic implant and may initiate or contribute to the cellular events that cause aseptic loosening by inhibiting bone formation. The effects on alkaline phosphatase and PGE2 release are dependent on the chemical composition of the particles, suggesting that both the type and concentration of wear debris at an implant site may be important in determining clinical outcome.     

Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin.

Bordetella pertussis and Bacillus anthracis produce extracytoplasmic adenylate cyclase toxins (AC toxins) with shared features including activation by calmodulin and the ability to enter target cells and catalyze intracellular cyclic AMP (cAMP) production from host ATP. The two AC toxins were evaluated for sensitivities to a series of inhibitors of known uptake mechanisms. Cytochalasin D, an inhibitor of microfilament function, abrogated the cAMP response to B. anthracis AC toxin (93%) but not the cAMP response elicited by B. pertussis AC toxin. B. anthracis-mediated intoxication of CHO cells was completely inhibited by ammonium chloride (30 mM) and chloroquine (0.1 mM), whereas the cAMP accumulation produced by B. pertussis AC toxin remained unchanged. The block of target cell intoxication by cytochalasin D could be bypassed when cells were first treated with anthrax AC toxin and then exposed to an acidic medium. These data indicate that despite enzymatic similarities, these two AC toxins intoxicate target cells by different mechanisms, with anthrax AC toxin entering by means of receptor-mediated endocytosis into acidic compartments and B. pertussis AC toxin using a separate, and as yet undefined, mechanism.     

Cyclic nucleotide signaling in Toxoplasma gondii bradyzoite differentiation

The ability of Toxoplasma gondii tachyzoites to differentiate into latent bradyzoite forms is essential for pathogenesis of clinical disease. We examined the effects of cyclic nucleotides on T. gondii bradyzoite differentiation in vitro. Differentiation of tachyzoites to bradyzoites was measured in an immunofluorescence assay using ME49 or its clonal derivative PLK, two well-characterized T. gondii strains. Treatment of human fibroblast cultures infected with T. gondii with 8-(4-chlorophenylthio)-cyclic GMP (CPT-cGMP), a membrane-permeable, nonhydrolyzable analogue of cGMP, resulted in an increased percentage of bradyzoite-positive vacuoles. Cyclic AMP (cAMP) also induced in vitro conversion of PLK, but the method of cAMP elevation was critical. Forskolin raises cAMP levels transiently and induced bradyzoites, whereas agents predicted to cause sustained elevation of cAMP were inhibitory to parasite conversion. Levels of cAMP were measured in host cells and extracellular tachyzoites. Forskolin, CPT-cGMP, and agents known to induce bradyzoite formation elevated cAMP in host cells and PLK parasites. These data suggest cyclic nucleotide signaling pathways are important in the stress-induced conversion of T. gondii tachyzoites to bradyzoites. Furthermore, because cAMP elevation was seen in PLK but not RH, a T. gondii strain that did not differentiate well in our assay, cAMP signaling within the parasite is likely to be critical.     

Stimulation of the differentiation of osteogenic rat bone marrow stromal cells by osteoblast cultures

The effect of conditioned medium (CM) from rat calvaria (RC) cel cultures on the growth and differentiation of osteogenic cells in rat bone marrow stromal cell (BMSC) cultures was investigated. Control cultures received either CM from periodontal ligament fibroblast cultures or fresh medium. RCCM stimulated the formation of nodules of bonelike tissue in bone marrow stromal cell cultures in a dose-dependent manner,and the maximal stimulation was associated with the osteoblast-enriched cell populations of the RC cultures. Ultrafiltration demonstrated that activity was confined to a CM fraction of 10- to 30-kilodalton molecular size. The activity was sensitive to boiling and trypsin treatments, but was not affected by neutralizing antibodies to transforming growth factor beta or insulin-like growth factor I or II. RCCM was found to initially increase the number and proportion of cells that expressed alkaline phosphatase activity, although the proportion of alkaline phosphatase-positive cells subsequently declined. These data were consistent with an initial stimulation of proliferation of a subpopulation of osteoprogenitor cells within the cultures, followed by their differentiation. The results suggest that mature osteoblasts may produce a paracrine growth factor that can stimulate the differentiation of osteoblasts from precursor cells.