ADAM12/syndecan-4 signaling promotes beta 1 integrin-dependent cell spreading through protein kinase Calpha and RhoA

The ADAMs (a disintegrin and metalloprotease) comprise a large family of multidomain proteins with cell-binding and metalloprotease activities. The ADAM12 cysteine-rich domain (rADAM12-cys) supports cell attachment using syndecan-4 as a primary cell surface receptor that subsequently triggers beta(1) integrin-dependent cell spreading, stress fiber assembly, and focal adhesion formation. This process contrasts with cell adhesion on fibronectin, which is integrin-initiated but syndecan-4-dependent. In the present study, we investigated ADAM12/syndecan-4 signaling leading to cell spreading and stress fiber formation. We demonstrate that syndecan-4, when present in significant amounts, promotes beta(1) integrin-dependent cell spreading and stress fiber formation in response to rADAM12-cys. A mutant form of syndecan-4 deficient in protein kinase C (PKC)alpha activation or a different member of the syndecan family, syndecan-2, was unable to promote cell spreading. GF109203X and G?6976, inhibitors of PKC, completely inhibited ADAM12/syndecan-4-induced cell spreading. Expression of syndecan-4, but not syn4DeltaI, resulted in the accumulation of activated beta(1) integrins at the cell periphery in Chinese hamster ovary beta1 cells as revealed by 12G10 staining. Further, expression of myristoylated, constitutively active PKCalpha resulted in beta(1) integrin-dependent cell spreading, but additional activation of RhoA was required to induce stress fiber formation. In summary, these data provide novel insights into syndecan-4 signaling. Syndecan-4 can promote cell spreading in a beta(1) integrin-dependent fashion through PKCalpha and RhoA, and PKCalpha and RhoA likely function in separate pathways.     

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Antisense DNA target sites can be selected by the accessibility of the mRNA target. It remains unknown whether a mRNA site that is accessible to an antisense DNA is also a good candidate target site for a siRNA. Here, we reported a parallel analysis of 12 pairs of antisense DNAs and siRNA duplexes for their potency to inhibit reporter luciferase activity in mammalian cells, both of the antisense DNA and siRNA agents in a pair being directed to same site in the mRNA. Five siRNAs and two antisense DNAs turned out to be effective, but the sites targeted by those effective siRNAs and antisense DNAs did not overlap. Our results indicated that effective antisense DNAs and siRNAs have different preferences for target sites in the mRNA.     

The STC-1 cells express functional orexin-A receptors coupled to CCK release

Orexins are newly discovered neuropeptides regulating feeding and vigilance and have been detected in neuroendocrine cells of the gut. Potential neuroendocrine functions of orexin are unknown. Therefore, the effects of orexin-A on the intestinal neuroendocrine cell line, STC-1, were investigated as a model system. RT-PCR demonstrated the presence of both OX(1) and OX(2) receptors. Stimulation with orexin-A produced a dose-dependent release of cholecystokinin (CCK), which was abolished by removal of extracellular Ca(2+) or the presence of the voltage-gated L-type Ca(2+)-channel blocker diltiazem (10 microM). Orexin-A (Ox-A) elevated intracellular Ca(2+), which was dependent on extracellular Ca(2+). Furthermore, orexin-A caused a membrane depolarization in the STC-1 cells. Ox-A neither elevated cAMP levels nor stimulated phosphoinositide turnover in these cells. These data demonstrate a functional orexin receptor in the STC-1 cell line. Ox-A produces CCK release in these cells, by a mechanism involving membrane depolarization and subsequently activation of L-type voltage-gated Ca(2+)-channels.     

Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.     

Regulation of TRKB surface expression by brain-derived neurotrophic factor and truncated TRKB isoforms

Brain-derived neurotrophic factor (BDNF) signaling through its receptor TRKB modulates survival, differentiation, and activity of neurons. BDNF activates TRKB on the cell surface, which leads to the initiation of intracellular signaling cascades and different biological responses in neurons. Neuronal activity has been shown to regulate TRKB levels on the plasma membrane of neurons, but little is known about other factors affecting TRKB surface expression levels. We report here that BDNF regulates the cell surface levels of transfected or endogenously expressed full-length TRKB, depending on the exposure time in neuroblastoma cells and primary hippocampal neurons. BDNF rapidly increases TRKB surface expression levels in seconds, whereas treatment of cells with BDNF for a longer time (minutes to hours) leads to decreased TRKB surface levels. Coexpression of the full-length TRKB together with the truncated TRKB.T1 isoform results in decreased levels of full-length TRKB on the cell surface. This effect is specific to the T1 isoform, because coexpression of a kinase-dead TRKB mutant or another kinase domain-lacking TRKB form, truncated T-Shc, leads to increased TRKB surface levels. Our results suggest that regulation of TRKB surface expression levels by different factors is tightly controlled by complex mechanisms in active neurons.     

X-ray structure and ligand binding study of a moth chemosensory protein

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. Such proteins, of M(r) 13,000, have been isolated from several sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. One of them, CSPMbraA6, a 112-amino acid antennal protein, has been expressed in large quantities and is soluble in the Escherichia coli periplasm. X-ray structure determination has been performed in parallel with ligand binding assays using tryptophan fluorescence quenching. The protein has overall dimensions of 25 x 30 x 32 A and exhibits a novel type of alpha-helical fold with six helices connected by alpha-alpha loops. A narrow channel extends within the protein hydrophobic core. Fluorescence quenching with brominated alkyl alcohols or fatty acids and modeling studies indicates that CSPMbraA6 is able to bind such compounds with C12-18 alkyl chains. These ubiquitous proteins might have the role of extracting hydrophobic linear compounds (pheromones, odors, or fatty acids) dispersed in the phospholipid membrane and transporting them to their receptor.     

Metabolic activation of A549 human airway epithelial cells by organic dust: a study based on microphysiometry

A Cytosensor microphysiometer, which measures extracellular acidification rate (ECAR), was used to study the early metabolic activation by organic dust from a swine confinement building in a human airway epithelial cell line, A549. The dust is known to cause an intense airway inflammatory reaction following inhalation in vivo and cytokine release in vitro. Dimethyl amiloride (DMA) was used to study sodium/proton exchanger (NHE) activity in cells growing at different cell densities. Exposing cells at low density to dust induced an initial release of acid not involving NHE, followed by a sustained DMA-sensitive NHE activation. In cells near high density, NHE was not activated during exposure resulting in a modest increase in ECAR. Exposing cells at high density resulted in a bi-phasic ECAR pattern; an initial increase in proton release followed by an inhibition of ECAR below baseline. Pretreatment with pertussis toxin (PTX), an inhibitor of receptor/G(i alpha)-coupled signal transductions did not affect ECAR in low and medium density cells, but abolished the inhibition of ECAR in high-density cells. The dust did not prevent forskolin-induced cAMP accumulation and PTX did not affect cAMP in near-confluent cells suggesting the PTX-effect to be cAMP-independent. The ECAR response to organic dust was similar to that of lipopolysaccharide (LPS) except for high-density cells where PTX did not influence the LPS-induced decrease in ECAR below baseline. In summary, the organic dust induces PTX-sensitive (cAMP independent) signalling in near-confluent A549 epithelial cells and, depending on cell density opposing effects on NHE activity during exposure.     

Influence of prior Cd(2+) exposure on the uptake of Cd(2+) and other elements in the phytochelatin-deficient mutant, cad1-3, of Arabidopsis thaliana

In order to test the potential effect of prior exposure to different Cd concentrations on Cd uptake and accumulation, plants of Arabidopsis thaliana, including a phytochelatin-deficient mutant, cad1-3, and the wild type, were compared. For Cd uptake experiments, plants were grown for 1 week in nutrient solution containing different Cd concentrations (0, 0.05, 0.1, 0.25, 0.5, and 1.0 microM Cd(NO(3))(2)). Thereafter they were subjected to 0.5 microM Cd labelled with (109)Cd for 2 h. Uptake experiments with (109)Cd showed that the phytochelatin-deficient mutant cad1-3, accumulated less Cd than the wild type. Both a lower proportion and lower total amount of absorbed Cd were translocated to the shoot in cad1-3 plants compared to wild-type plants. Cadmium exposure also influenced the amounts of nutrients found, whereby after exposure to high Cd concentrations (0.5, 1.0 microM) during growth, cad1-3 roots contained less Fe, K, Mg, P, and S compared to roots of the wild type. In cad1-3 these elements decreased with increasing Cd concentration. The total Cd content in roots and shoots increased significantly with increasing Cd concentration during growth, although the increase was much less in cad1-3 plants. In time-dependent experiments of Cd uptake carried out between 15 and 120 min on plants not previously exposed to Cd, no significant difference in Cd accumulation between the mutant and wild type were found, although a smaller amount of Cd was translocated to the shoot in cad1-3 plants. The possibility that the differences in Cd accumulation in mutant and wild-type lines may be due to the cytosolic Cd regulation, which is inhibited by the complexation of Cd by phytochelatins, is discussed.