Antagonistic effects of TGFbeta1 and BMP-6 on skin keratinocyte differentiation

Several members of the transforming growth factor beta (TGFbeta) superfamily are expressed in the developing murine epidermis. Among these are TGFbeta1, which is found in the basal layer, and bone morphogenetic protein (BMP)-6, located in the suprabasal layers. Although the role of TGFbeta in cell growth has been studied extensively, little is known about the effects of these factors on keratinocyte differentiation. This study demonstrates that BMP-6 acts to positively regulate the differentiation of primary skin keratinocytes grown in culture. In contrast, TGFbeta1 antagonizes keratinocyte differentiation blocking the upregulation of keratin markers by BMP-6. We show that the effects of BMP-6 on expression of keratin 1 (K1), a marker of differentiation, requires signaling through the Smad pathway. In addition, regulation of K1 levels by BMP-6 is modulated by the SEK signaling pathway. This suggests that regulation of keratinocyte differentiation by BMP-6 involves multiple signaling systems.     

Overexpression of a kinase-deficient transforming growth factor-beta type II receptor in mouse mammary stroma results in increased epithelial branching

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through heteromeric type I and type II serine/threonine kinase receptors. Transgenic mice that overexpress a dominant-negative mutation of the TGF-beta type II receptor (DNIIR) under the control of a metallothionein-derived promoter (MT-DNIIR) were used to determine the role of endogenous TGF-betas in the developing mammary gland. The expression of the dominant-negative receptor was induced with zinc and was primarily localized to the stroma underlying the ductal epithelium in the mammary glands of virgin transgenic mice from two separate mouse lines. In MT-DNIIR virgin females treated with zinc, there was an increase in lateral branching of the ductal epithelium. We tested the hypothesis that expression of the dominant-negative receptor may alter expression of genes that are expressed in the stroma and regulated by TGF-betas, potentially resulting in the increased lateral branching seen in the MT-DNIIR mammary glands. The expression of hepatocyte growth factor mRNA was increased in mammary glands from transgenic animals relative to the wild-type controls, suggesting that this factor may play a role in TGF-beta-mediated regulation of lateral branching. Loss of responsiveness to TGF-betas in the mammary stroma resulted in increased branching in mammary epithelium, suggesting that TGF-betas play an important role in the stromal-epithelial interactions required for branching morphogenesis.     

Morphometric prognostic index in breast cancer

OBJECTIVE: To assess the ability of the morphometric prognostic index (MPI) in predicting clinical outcome in a group of breast cancer patients with short-term follow-up and to assess the relationship between MPI and other prognosticators. STUDY DESIGN: The study group consisted of 63 cases of breast cancer. Follow-up data were available for 48 patients. MPI values were calculated, and degree of nuclear and tubular differentiation was investigated in each tumor. S-phase fraction (SPF), estrogen and progesterone receptors were also studied. RESULTS: The group of patients with MPI values < 0.60 had percent values of disease-free survival significantly higher than did those with MPI values > or = 0.60. Furthermore, significant direct correlations were found between MPI and degree of nuclear atypia and between MPI and SPF. Significant inverse relationships were found between MPI and tumor progesterone receptor levels and between MPI and degree of histologic tubular differentiation. CONCLUSION: The validity of MPI as a prognosticator in breast cancer was confirmed, even in a limited number of patients observed in short-term follow-up. MPI seems to be a reliable and economical prognosticator in selecting breast cancer patients for adjuvant chemotherapy.     

The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.     

Conductive properties and gating of channels formed by syringopeptin 25A, a bioactive lipodepsipeptide from Pseudomonas syringae pv. syringae, in planar lipid membranes

Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.     

Prolactin and transforming growth factor-beta signaling exert opposing effects on mammary gland morphogenesis, involution, and the Akt-forkhead pathway

Both prolactin (PRL) and TGF-beta regulate cell survival in mammary epithelial cells, but their mechanisms of interactions are not known. In primary mammary epithelial cells and the HC11 mouse mammary epithelial cell line, PRL prevented TGF-beta-induced apoptosis, as measured by terminal deoxynucleotidyltransferase dUTP nick-end labeling staining and caspase-3 activation. This effect depended on phosphatidyl inositol triphosphate kinase (PI3K). PI3K activates a downstream serine/threonine kinase, Akt; therefore, we investigated the role of Akt in the interaction between PRL and TGF-beta signaling. Akt activity was inhibited by TGF-beta over a 20- to 60-min time course. In TGF-beta-treated cells, PRL disinhibited Akt in a PI3K-dependent manner. Expression of dominant negative Akt blocked the protective effect of PRL in TGF-beta-induced apoptosis. Transgenic mice overexpressing a dominant-negative TGF-beta type II receptor (DNIIR) in the mammary epithelium undergo hyperplastic alveolar development, and this effect was PRL dependent. Involution in response to teat sealing was slowed by overexpression of DNIIR; furthermore, Akt and forkhead phosphorylation increased in the sealed mammary glands of DNIIR mice. Thus, Akt appears to be an essential component of the interaction between PRL and TGF-beta signaling in mammary epithelial cells both in vitro and in vivo.     

Changes in Abscisic Acid During Leaf Yellowing of Cut Stock Flowers

Leaf pigments, such as chlorophyll and carotenoids, are essential plant molecules. They provide carbohydrates and energy during all plant life. Leaf pigments are also important parameters of decorative plants, such as floriculture items, cut foliage and flowers. Leaf yellowing is a form of senescence caused by an internal hormone imbalance, such as a lack of cytokinins. The aim of this study was to investigate the changes in total carotenoids and endogenous ABA in cut flower stock leaves during post-harvest life. The effect of pulse treatment with 5 or 10 M thidiazuron (TDZ), 150 mg l^–1 8-hydroxyquinoline sulphate (8-HQS) and combinations of TDZ with 8-HQS on total carotenoids and ABA concentration was also investigated. Results showed that total carotenoids drastically decreased from 1548 g cm^–2, until reaching 565 g cm_–2 at the end of vase life. Endogenous ABA strongly increased at the same time, going from 167 ng g^–1 DW at the beginning of the experiment to 1322 ng g^–1 DW at the end of vase life. The TDZ inhibited carotenoid degradation, but did not affect the ABA concentration, while the 8-HQS did not prevent carotenoid degradation and the ABA concentration was only slightly affected. ABA seems to be a secondary senescence bio-product that may have a synergic effect with other senescence inducers dramatically accelerating leaf senescence.     

Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.     

A novel mechanism of pore formation: membrane penetration by the N-terminal amphipathic region of equinatoxin

Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10-28 are organized as an alpha-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation.