Significance of N-methyl-D-aspartate (NMDA) receptor-mediated signaling in human keratinocytes

Increasing data suggest that glutamate might act as a cell-signaling molecule in non-neuronal tissues such as the skin. Here we demonstrate the presence of functional N-methyl-D-aspartate (NMDA)-type glutamate receptors in human keratinocytes. NMDA receptor expression strongly reflects the degree of cell-to-cell contact. Wounding polarizes the expression of NMDA receptors in keratinocytes involved in re-epithelialization, and the process of re-epithelialization is inhibited by NMDA receptor activation. We also demonstrate that squamous cell carcinomas lack NMDA receptors. Our data suggest that Ca2+ entry through NMDA receptors influences the cycle of keratinocyte proliferation, differentiation, and migration during epithelialization. Moreover, NMDA receptor activation might play a role in contact-mediated inhibition of growth, a process that is absent during neoplastic pathology. This receptor may serve as a pharmacological target for modulating keratinocyte behavior and treating cutaneous disorders.     

Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity

In response to ambient hypertonicity, TonEBP (tonicity-responsive enhancer binding protein) stimulates certain genes including those encoding cytokines, transporters for organic solutes, and a molecular chaperone. TonEBP is regulated in a bidirectional manner, upregulated by an increase in ambient tonicity while downregulated by a decrease. To investigate the role of intracellular ionic strength in the activity of TonEBP, we subjected Madin-Darby canine kidney cells to a variety of conditions. Electron microprobe analysis was performed to measure intracellular electrolytes. Under conditions in which changes in cell volume were similar, TonEBP activity correlated with the intracellular ionic strength regardless of the external tonicity. On the other hand, inhibition of the Na+/K+-ATPase and high external K+ concentration led to a decreased activity of TonEBP despite a marked increase in the intracellular ionic strength. Because isotonic swelling is known to occur under these conditions, these data suggest that dilution of the cytoplasmic constituents inhibits the activity of TonEBP. We conclude that intracellular ionic strength and water content are major factors that determine the activity of TonEBP.     

Decreased calretinin expression in cerebellar granule cells in the leaner mouse

We investigated calretinin expression in cerebellar granule cells of 30-day-old leaner mice to understand possible changes in calcium homeostasis due to the calcium channel mutation that these mice carry. Quantitative in situ hybridization histochemistry showed decreased calretinin mRNA expression in the leaner cerebellum. Immunohistochemical staining also revealed decreased calretinin immunoreactivity in the leaner cerebellum. To exclude the effect of granule cell loss that occurs in the leaner mouse when comparing cerebellar calretinin expression, the number of granule cells per unit area in the cerebellum was compared to the wild-type cerebellum. Granule cell counts per unit area of cerebellum revealed similar numbers of granule cells present in wild-type and leaner mice. Laser capture microdissection (LCM) was employed to obtain an equal number of granule cells from wild-type and leaner mice. Western blot analysis with LCM-procured cerebellar granule cells showed decreased calretinin expression in leaner granule cells. These results indicate that there is an absolute decrease in calretinin expression in leaner granule cells even when granule cell loss is taken into account. Decreased calretinin expression in leaner granule cells may contribute to altered calcium buffering capacity. This alteration could be an adaptive change due to the calcium channel dysfunction, and may result in abnormal neuronal excitability and gene expression.     

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.     

Human antibodies to phosphocholine. IgG anti-PC antibodies express restricted numbers of V and C regions

We examined the IgG subclass composition and isoelectric focusing (IEF) spectrotype pattern of naturally occurring human IgG antibodies that bind phosphocholine (PC) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isotype of these IgG anti-PC antibodies was primarily IgG2. However, serum from some individuals contained significant amounts of IgG1 and IgG3 anti-PC antibodies. We also found that in individual sera, anti-PC antibodies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IEF pattern of these antibodies were for the most part unique for each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH bound PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumococcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG1 subclass. Taken together, these findings demonstrate that within individual sera, human antibodies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate.     

Ecophysiology of selected tree species in different plant communities at the periphery of the Atlantic Forest of SE Brazil I. Performance of three different species of Clusia in an array of plant communities

Three species of Clusia, namely two CAM-species (C. hilariana Schlecht. and C. fluminensis Planch. et Triana) and a C₃-species (C. parviflora Saldanha et Engl.) were studied in different plant communities at the periphery of the Atlantic Forest in the state of Rio de Janeiro, Brazil. The sites chosen were an inselberg (C. parviflora), a range of wet restinga – dry restinga – semideciduous dry forest (C. fluminensis), and a gradient from the sea shore inland with a first sand dune beach ridge – a dry forest – a second beach ridge (C. hilariana). Analyses comprised C and N contents, soluble carbohydrates, soluble nonprotein N compounds and carbon isotope ratios (¹³C) in leaves, roots, phloem and wood. Photosynthetic performance was assessed by chlorophyll fluorescence with measurements of instant photosynthetic yield as well as light dependence and potential quantum-use efficiency of photosystem II.The data allow, first, to discuss differences between the ecophysiological performance of C₃- and CAM-species of Clusia. The C₃-species, C. parvifolia, had an overall weaker performance than the two CAM-species, where, however, the effects of mode of photosynthesis may have been overlaid by site conditions. Second, it was studied whether ecophysiological performance relates to patterns of local abundance, which was confirmed by showing that the dominant Clusia species of the restingas, C. hilariana, showed the strongest performance overall.Finally, it was studied whether the ecophysiological performance varied in response to site-dependent gradients of environmental water relations, which was confirmed for functions such as photosynthetic capacity, photoinhibition and solute accumulation of C. hilariana and C. fluminensis in relation to moisture of sites.     

OsGAP1 functions as a positive regulator of OsRab11-mediated TGN to PM or vacuole trafficking

The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase-green fluorescent protein (GFP) and Ca2+-ATPase8-GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)-GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11.