Ligand-dependent and -independent interactions with the transforming growth factor type II and I receptor subunits reside in the aminoterminal portion of the ectodomain of the type III subunit

Summary The type III receptor for transforming growth factor beta (TGFβ), which exhibits no kinase activity, binds TGFβ1 and TGFβ2 and is involved in assembly and activity of the multi-subunit TGFβ signal transduction complex. Recently we showed that TGFβ receptor type III (TβRIII) can participate in a complex composed of the dimeric TGFβ ligand and a type III, II, and I receptor subunit. The interaction of the TβRIII subunit with TβRII is TGFβ-dependent, whereas interaction with TβRI is TGFβ-independent. Here we use coexpression of the three types of TGFβ receptors in baculoviral-infected insect cells to determine which parts of the unglycosylated TβRIII receptor participate in the binding of TGFβ, the TGFβ-dependent interaction with TβRII and the TGFβ-independent interaction with TβRI. The results suggest that the first 500 amino acid residues in the aminoterminal portion of TβRIII exhibit all three properties.     

Effect of bile acids on the uptake of irinotecan and its active metabolite, SN-38, by intestinal cells

The intestinal transport of irinotecan (CPT-11) and its active metabolite, SN-38, has been previously reported (K. Kobayashi et al., Int. J. Cancer, 83 (1999) 491-496). In the present study, the effect of the two major primary bile acids, cholic acid (CA) and taurocholic acid (TCA), on the uptake of CPT-11 and SN-38 by hamster intestinal epithelial cells was investigated. These two bile acids at concentrations up to 200 microM did not directly alter the cellular uptake of CPT-11 and SN-38. However, under physiologically acidic intestinal pH conditions, micelle formation induced by 20 mM TCA significantly reduced the cellular uptake of CPT-11 and SN-38 by 60% and 80%, respectively.     

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.     

Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes

Animals as well as plants defend themselves against invading pathogenic microorganisms utilizing cationic antimicrobial peptides, which rapidly kill various microbes without exerting toxicity against the host. Physicochemical peptide-lipid interactions provide attractive mechanisms for innate immunity. Many of these peptides form cationic amphipathic secondary structures, typically alpha-helices and beta-sheets, which can selectively interact with anionic bacterial membranes by the aid of electrostatic interactions. Rapid, peptide-induced membrane permeabilization is an effective mechanism of antimicrobial action. This review article summarizes interactions with lipid bilayers of magainins (alpha-helix) and tachyplesins (beta-sheet) discovered in frog skin and horseshoe crab hemolymph, respectively, as archetypes, emphasizing that the mode of interaction is strongly dependent on the physicochemical properties not only of the peptide, but also of the target membrane.     

Activation of protein kinase B induced by H(2)O(2) and heat shock through distinct mechanisms dependent and independent of phosphatidylinositol 3-kinase

Protein kinase B (PKB) is a downstream target of phosphatidylinositol (PI) 3-kinase in the signaling pathway of growth factors, and is activated by cellular stress such as H(2)O(2) and heat shock. To study the mechanism of the stress-induced activation of PKB, PI 3-kinase products were measured in stress-stimulated cells. Both PI 3,4-bisphosphate and PI 3,4, 5-trisphosphate increased in H(2)O(2)-treated cells, and the elevation of these phospholipids and activation of PKB were concurrently blocked by wortmannin, a potent inhibitor of PI 3-kinase. In heat-shocked cells, the level of PI 3,4-bisphosphate did not change while that of PI 3,4,5-trisphosphate increased slightly, and an association between PKB molecules was observed. Two active PKB fractions, presumably monomeric and oligomeric forms, were resolved from heat-shocked cells by gel filtration column chromatography. Activation of the former was suppressed by pretreatment with wortmannin, whereas the generation and activation of the latter were not blocked by the PI 3-kinase inhibitor. Only the monomeric form, but not the oligomeric form, was recovered from H(2)O(2)-treated cells, and its activation was prevented by wortmannin. These results indicate that PKB is activated by two distinct mechanisms that are dependent and independent of PI 3-kinase in stress-stimulated cells.     

A one-dimensional unsteady separable and reattachable flow model for collapsible tube-flow analysis

Taking into account both flow separation and reattachment observed in available experimental results on flows in a quasi-two-dimensional channel, we present a one-dimensional unsteady flow model, which is applicable to a flow in a collapsible tube. The flow model has been derived from the two-dimensional Navier-Stokes equations by introducing the concept of a dividing streamline, which divides a separated flow into a jet and a dead-water zone. We also present a criterion for the determination of a separation point. Numerical results show that the locations of the predicted separation points agree well with the experimental data. The predicted static pressure of the separated flow is almost constant downstream of the separation point and increases quickly just before the reattachment point as observed in the experiment. Finally, using the present flow model and the separation criterion, we examine the oscillatory behavior of an unsteady flow in a symmetric channel whose walls move sinusoidally.     

Transforming growth factor-beta1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cells

The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system.     

Effects of gender on kidney function in magnesium-deficient rats

This study investigated the gender differences in the kidney function of magnesium (Mg)-deficient rats. Male and female rats were fed a control diet or a Mg-deficient diet for 21 d. Mg-deficient diet had no significant effect on kidney calcium (Ca) or phosphorus (P) concentration in male rats, while Ca and P concentrations in female rats were significantly higher in Mg-deficient rats than in the control rats. With regard to indicators of kidney function, no significant differences in creatinine clearance and serum urea nitrogen concentration were observed among the groups. Serum albumin concentrations were significantly lower in rats fed the Mg-deficient diet than in rats fed the control diet. In both sexes, urinary albumin excretion was significantly higher in rats fed the Mg-deficient diet than in rats fed the control diet. Gender differences had no significant influence on creatinine clearance, serum urea nitrogen concentration, serum albumin concentration and urinary albumin excretion. These results suggest that gender differences have no effect on kidney function in Mg-deficient rats under the condition used.     

In vitro differentiation from naive to mature E-selectin binding CD4 T cells: acquisition of skin-homing properties occurs independently of cutaneous lymphocyte antigen expression

We previously showed that skin-homing CD4 T cells in peripheral blood can be subdivided into three populations on the basis of the expression pattern of the cutaneous lymphocyte Ag (CLA) and fucosyltransferase VII (FucT-VII): FucT-VII(+)CLA(-), FucT-VII(+)CLA(+), and FucT-VII(-)CLA(+). In view of the known late appearance of CLA during T cell differentiation, T cells programmed to attain skin-homing properties may start to generate E-selectin-binding epitopes at early stages of differentiation before induction of CLA expression. To this end, the in vitro differentiation from naive to CLA(+) memory T cells was followed after activation with anti-CD3 mAb. Here we demonstrate that naive skin-homing CD4 T cell precursors undergo a linear differentiation process from the FucT-VII(+)CLA(-) phenotype to the FucT-VII(+)CLA(+) phenotype and eventually to the FucT-VII(-)CLA(+) phenotype. The appearance of the FucT-VII(+)CLA(-) subset coincided with or could be immediately followed by the generation of E-selectin binding epitopes, and even after E-selectin-binding epitopes were no longer detectable, CLA remained expressed for prolonged periods of time, suggesting that induction of functional E-selectin ligands depends primarily on the expression of FucT-VII, but not CLA. Immunofluorescence and confocal microscopy studies of these T cells confirm that most E-selectin ligands were found independently of CLA expression.