IgE receptor type I-dependent regulation of a Rab3D-associated kinase: a possible link in the calcium-dependent assembly of SNARE complexes

Following activation through high affinity IgE receptors (FcepsilonRI), mast cells release, within a few minutes, their granule content of inflammatory and allergic mediators. FcepsilonRI-induced degranulation is a SNARE (soluble N-ethylmaleimide attachment protein receptors)-dependent fusion process. It is regulated by Rab3D, a subfamily member of Rab GTPases. Evidence exists showing that Rab3 action is calcium-regulated although the molecular mechanisms remain unclear. To obtain an understanding of Rab3D function we have searched for Rab3D-associated effectors that respond to allergic triggering through FcepsilonRI. Using the RBL-2H3 mast cell line we detected a Ser/Thr kinase activity, termed here Rak3D (from Rab3D-associated kinase), because it was specifically co-immunoprecipitated with anti-Rab3D antibody. Rak3D activity, as measured by its auto- or transphosphorylation, was maximal in resting cells and decreased upon stimulation. The down-regulation of the observed activity was blocked with EGTA, but not with other degranulation inhibitors, suggesting that its activity functions downstream of calcium influx. We found that Rak3D phosphorylates the NH(2)-terminal regulatory domain of the t-SNARE syntaxin 4, but not syntaxin 2 or 3. The phosphorylation of syntaxin 4 decreased its binding to its partner SNAP23. Thus, we propose a novel phosphorylation-dependent mechanism by which Rab3D controls SNARE assembly in a calcium-dependent manner.     

Regulation of skeletal muscle tension redevelopment by troponin C constructs with different Ca2+ affinities

In maximally activated skinned fibers, the rate of tension redevelopment (ktr) following a rapid release and restretch is determined by the maximal rate of cross-bridge cycling. During submaximal Ca2+ activations, however, ktr regulation varies with thin filament dynamics. Thus, decreasing the rate of Ca2+ dissociation from TnC produces a higher ktr value at a given tension level (P), especially in the [Ca2+] range that yields less than 50% of maximal tension (Po). In this study, native rabbit TnC was replaced with chicken recombinant TnC, either wild-type (rTnC) or mutant (NHdel), with decreased Ca2+ affinity and an increased Ca2+ dissociation rate (koff). Despite marked differences in Ca2+ sensitivity (>0.5 DeltapCa50), fibers reconstituted with either of the recombinant proteins exhibited similar ktr versus tension profiles, with ktr low (1-2 s-1) and constant up to approximately 50% Po, then rising sharply to a maximum (16 +/- 0.8 s-1) in fully activated fibers. This behavior is predicted by a four-state model based on coupling between cross-bridge cycling and thin filament regulation, where Ca2+ directly affects only individual thin filament regulatory units. These data and model simulations confirm that the range of ktr values obtained with varying Ca2+ can be regulated by a rate-limiting thin filament process.     

The MMP/TIMP system in the nervous system

The matrix metalloproteinases (MMP) belong to a growing family of secreted or membrane-bound (MT-MMP) enzymes that cleave protein components of the extracellular matrix and bioactive factors involved in intercellular signaling. MMP activity is counterbalanced by their four physiological inhibitors, the tissue inhibitors of MMP (TIMPs). Together, MMP and TIMP control cell-cell and cell-matrix interactions associated with physiological processes. However, the breakdown of the protease-inhibitor balance may lead to the loss of tissue homeostasis and the development of degenerative and tumorigenic processes in various tissues. The emerging idea is that the MMP/TIMP system also plays a major role in the pathology and physiology of the nervous system and that mastering MMP activity will set the basis for new and more efficient therapeutic strategies against nervous system disorders.     

Ascorbate distribution during hibernation is independent of ascorbate redox state

Distribution of ascorbate into tissues is an essential process in ascorbate antioxidant defense. Hibernating animals are studied as a model of tolerance to ischemia-reperfusion because of their tolerance to fluctuations in blood flow associated with prolonged torpor and periodic arousal episodes. Throughout hibernation, plasma ascorbate concentration ([Asc](p)) repetitively increases during torpor, then falls during periodic arousal bouts. We previously proposed that high [Asc](p) provides a ready source of antioxidant protection for distribution to the central nervous system and peripheral tissues during arousal. Here we tested whether deliberate oxidation of plasma ascorbate by intravenous administration of ascorbate oxidase (AO), prior to arousal, compromised tissue levels of ascorbate or the other water-soluble antioxidants, glutathione (GSH) and urate. Although AO decreased [Asc](p) to below the level of detection during torpor and after arousal, ascorbate oxidation did not decrease post-arousal tissue levels of reduced ascorbate, glutathione, or urate in any tissue examined, except liver. The data imply that ascorbate is taken up equally well into brain and other tissues as either ascorbate or its oxidized product dehydroascorbate, with subsequent intracellular reduction of dehydroascorbate. Lack of effect of ascorbate oxidation on tissue levels of GSH or urate indicates that dehydroascorbate uptake and reduction do not compromise tissue concentrations of these other water-soluble antioxidants. Thus, we show equal availability of reduced and oxidized plasma ascorbate during metabolically demanding thermogenesis and reperfusion associated with arousal from hibernation.     

Black flower coloration in wild Lisianthius nigrescens: its chemistry and ecological consequences

The major pigments responsible for the flower color within the black flowered Gentianaceae, Lisianthius nigrescens, were characterized by HPLC and chemical analyses HPLC analysis showed one major and one minor anthocyanin and 3 major and 3 minor flavone glycosides. The anthocyanins [delphinidin-3-O-rhamnol(1-6)galactoside and its 5-O-glucoside] comprised an extraordinary 24% of the dry weight of wild collected L. nigrescens corallas, and were accompanied in a 1:1 ratio by a range of apigenin and luteolin 8-C-glucosides and their 7-O-methyl ethers. The high levels of anthocyanins and flavones (and their co-pigmentation) is thought to account for the almost complete absorption of both UV and visible wavebands observed by reflectance photography.     

Arbuscular mycorrhizal fungi colonize decomposing leaves of<Emphasis Type="Italic"> Myrica parvifolia</Emphasis>,<Emphasis Type="Italic"> M. pubescens</Emphasis> and<Emphasis Type="Italic"> Paepalanthus</Emphasis> sp.

Hyphae and vesicles of arbuscular mycorrhizal fungi (AMF) were found within the decomposing leaves of Myrica parvifolia, M. pubescens and Paepalanthus sp. at three montane sites in Colombia. Hyphae, vesicles, and arbuscule-like structures were also found within scale-like leaves of the rhizomes of Paepalanthus sp. The litter found in the vicinity of the roots was divided into three decomposition layers. The highest AMF colonization occurred in the most decomposed leaves, which were in close association with roots. In contrast, there were no differences in AMF colonization of roots present in the different decomposition layers. Colonization of decomposing leaves by AMF did not differ between the two closely related species M. parvifolia and M. pubescens, nor between two sites (Guatavita and Zipacón, Colombia) differing in soil fertility. Occurrence of vesicles in decomposing leaves was correlated with abundant AMF extraradical hyphae among the leaves. We propose that AMF enter decomposing leaves mechanically through vascular tissue. As a consequence, AMF are well positioned to obtain and efficiently recycle mineral nutrients released by decomposer microorganisms before their loss by leaching or immobilization in soil.     

More is not necessarily better: prozone-like effects in passive immunization with IgG

Despite a century of study, the relationship between Ag-specific Ig concentration and protection remains poorly understood for the majority of pathogens. In certain conditions, administration of high Ab doses before challenge with an infectious agent can be less effective than smaller Ab doses, a phenomenon which is consistent with a prozone-like effect. In this study, the relationship between IgG1, IgG2a, IgG2b, and IgG3 dose, infective inocula, and protection was investigated in a mouse model of Cryptococcus neoformans infection. The activity of each IgG subclass ranged from protective to disease-enhancing depending on both the Ab dose and infective inocula used. Enhanced dissemination to the brain was observed in mice given a high IgG2a dose and a relatively low inoculum. Ab administration had immunomodulatory effects, with cytokine expression in lung, brain, and spleen varying as a function of the infective inoculum Ab dose and IgG subclass. In vitro studies did not predict or explain the mechanism of in vivo prozone-like effects, because all isotypes were opsonic and elicited NO release from macrophages. IgG2a was most efficient in inducing a macrophage oxidative burst. These results reveal that an individual Ab can be protective, nonprotective, or disease-enhancing depending on its concentration relative to a challenge inoculum. Our findings have implications for the potential contribution of Ab responses to defense against microbial diseases because Ab-mediated immunity may be protective, nonprotective, or even deleterious to the host.     

Pure partial trisomy 6p due to a familial insertion (16;6)(p12;p21.2p23)

There have only been eight patients with 6p pure trisomy involving different segments: four cases resulted from a translocation or insertion and four were due to an intrachromosomal duplication. We report here the first postnatally ascertained patient with a pure 6p partial trisomy due to an interchromosomal insertion (16;6)(p12;p21.2p23)mat. This rearrangement was confirmed by fluorescent in situ hybridization (FISH) with whole chromosome 6 and 16 painting probes. The clinical findings in the present patient were similar to those observed in previous cases, including craniofacial dysmorphism, minor anomalies, and lack of severe anatomical defects; yet, the unspecificity of many of these features prevented us from delineating the 6p pure trisomy syndrome.