Loss of RAB-3/A in Caenorhabditis elegans and the mouse affects behavioral response to ethanol

The mechanisms by which ethanol induces changes in behavior are not well understood. Here, we show that Caenorhabditis elegans loss-of-function mutations in the synaptic vesicle-associated RAB-3 protein and its guanosine triphosphate exchange factor AEX-3 confer resistance to the acute locomotor effects of ethanol. Similarly, mice lacking one or both copies of Rab3A are resistant to the ataxic and sedative effects of ethanol, and Rab3A haploinsufficiency increases voluntary ethanol consumption. These data suggest a conserved role of RAB-3-/RAB3A-regulated neurotransmitter release in ethanol-related behaviors.     

Cytoplasmic truncation of glycoprotein Ib alpha weakens its interaction with von Willebrand factor and impairs cell adhesion

The interaction of the platelet glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib-IX-V complex, the recognition of VWF appears to be mediated entirely by GP Ibalpha, the largest of four GP Ib-IX-V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibalpha in the GP Ib-IX-VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibbeta, GP IX, and either wild-type GP Ibalpha or GP Ibalpha mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibalpha polypeptides with truncations of 6-82 amino acids rolled faster than cells expressing wild-type GP Ibalpha. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibalpha with longer truncations. The rolling velocity of cells expressing truncated GP Ibalpha mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibalpha-VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibalpha-VWF bonds.     

Kinetics of GPIbalpha-vWF-A1 tether bond under flow: effect of GPIbalpha mutations on the association and dissociation rates

The interaction between platelet glycoprotein (GP) Ib-IX-V complex and von Willebrand factor (vWF) is the first step of the hemostatic response to vessel injury. In platelet-type von Willebrand disease, two mutations, G233V and M239V, have been described within the Cys209-Cys248 disulfide loop of GPIbalpha that compromise hemostasis by increasing the affinity for vWF. We have earlier shown that converting other residues in this region to valine alters the affinity of GPIbalpha for vWF, with mutations K237V and Q232V, respectively, showing the greatest increase and decrease in affinity. Here, we investigated further the effect of these two mutations on the kinetics of the GPIbalpha interaction with the vWF-A1 domain under dynamic flow conditions. We measured the cellular on- and off-rate constants of Chinese hamster ovary cells expressing GPIb-IX complexes containing wild-type or mutant GPIbalpha interacting with vWF-A1-coated surfaces at different shear stresses. We found that the gain-of-function mutant, K237V, rolled very slowly and continuously on vWF-A1 surface while the loss-of-function mutant, Q232V, showed fast, saltatory movement compared to the wild-type (WT). The off-rate constants, calculated based on the analysis of lifetimes of transient tethers formed on surfaces coated with limiting densities of vWF-A1, revealed that the Q232V and K237V dissociated 1.25-fold faster and 2.2-fold slower than the WT. The cellular on-rate constant of WT, measured in terms of tethering frequency, was threefold more and threefold less than Q232V and K237V, respectively. Thus, the gain- and loss-of-function mutations in GPIbalpha affect both the association and dissociation kinetics of the GPIbalpha-vWF-A1 bond. These findings are in contrast to the functionally similar selectin bonds where some of the mutations have been reported to affect only the dissociation rate.     

A study of the spectral and redox properties and covalent flavinylation of the flavoprotein component of p-cresol methylhydroxylase reconstituted with FAD analogues

The spectral and redox properties are described for the wild-type and Y384F mutant forms of the flavoprotein component (PchF) of flavocytochrome, p-cresol methylhydroxylase (PCMH), and cytochrome-free PchF that harbor FAD analogues. The analogues are iso-FAD (8-demethyl-6-methyl-FAD), 6-amino-FAD (6-NH(2)-FAD), 6-bromo-FAD (6-Br-FAD), 8-nor-8-chloro-FAD (8-Cl-FAD), and 5-deaza-5-carba-FAD (5-deaza-FAD). All of the analogues bound noncovalently and stoichiometrically to cytochrome-free apo-PchF, and the resulting holoproteins had high affinity for the cytochrome subunit, PchC. Noncovalently bound FAD, 6-Br-FAD, or 6-NH(2)-FAD can be induced to bind covalently by exposing holo-PchF to PchC. The rate of this process and the redox potential of the noncovalently bound flavin may be correlated. In addition, the redox potential of each FAD analogue was higher when it was covalently bound than when noncovalently bound to PchF. Furthermore, the potential of a covalently bound or noncovalently bound FAD analogue increased on association of the corresponding holo-PchF with PchC, and the activity increased as the flavin's redox potential increased. It was discovered also that 4-hydroxybenzaldehyde, the final p-cresol oxidation product, is an efficient competitive inhibitor for substrate oxidation by PchF since it binds tightly to this protein when the flavin is oxidized, although it binds more loosely to the enzyme with reduced flavin. Finally, the energies of the charge-transfer bands for the interaction of bound flavin analogues with 4-Br-phenol (a substrate mimic) increased as the potential decreases, although a simple global correlation was not seen. This is the case because the energy is also a function of the redox properties of the bound mimic. The implications of these findings to covalent flavinylation and catalysis are discussed.     

Critical role for IL-13 in the development of allergen-induced airway hyperreactivity

Airway hyperresponsiveness to a variety of specific and nonspecific stimuli is a cardinal feature of asthma, which affects nearly 10% of the population in industrialized countries. Eosinophilic pulmonary inflammation, eosinophil-derived products, as well as Th2 cytokines IL-13, IL-4, and IL-5, have been associated with the development of airway hyperreactivity (AHR), but the specific immunological basis underlying the development of AHR remains controversial. Herein we show that mice with targeted deletion of IL-13 failed to develop allergen-induced AHR, despite the presence of vigorous Th2-biased, eosinophilic pulmonary inflammation. However, AHR was restored in IL-13(-/-) mice by the administration of recombinant IL-13. Moreover, adoptive transfer of OVA-specific Th2 cells generated from TCR-transgenic IL-13(-/-) mice failed to induce AHR in recipient SCID mice, although such IL-13(-/-) Th2 cells produced high levels of IL-4 and IL-5 and induced significant airway inflammation. These studies definitively demonstrate that IL-13 is necessary and sufficient for the induction of AHR and that eosinophilic airway inflammation in the absence of IL-13 is inadequate for the induction of AHR. Therefore, treatment of human asthma with antagonists of IL-13 may be very effective.     

Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region

The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator-dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ib alpha antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ib alpha for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction.     

Calcium‐ and potassium‐permeable plasma membrane transporters are activated by copper in Arabidopsis root tips: linking copper transport with cytosolic hydroxyl radical production

Transition metals such as copper can interact with ascorbate or hydrogen peroxide to form highly reactive hydroxyl radicals (OH^?), with numerous implications to membrane transport activity and cell metabolism. So far, such interaction was described for extracellular (apoplastic) space but not cytosol. Here, a range of advanced electrophysiological and imaging techniques were applied to Arabidopsis thaliana plants differing in their copper‐transport activity: Col‐0, high‐affinity copper transporter COPT1‐overexpressing (C1^OE) seedlings, and T‐DNA COPT1 insertion mutant (copt1). Low Cu concentrations (10 µm ) stimulated a dose‐dependent Gd³⁺ and verapamil sensitive net Ca²⁺ influx in the root apex but not in mature zone. C1^OE also showed a fivefold higher Cu‐induced K⁺ efflux at the root tip level compared with Col‐0, and a reduction in basal peroxide accumulation at the root tip after copper exposure. Copper caused membrane disruptions of the root apex in C1^OE seedlings but not in copt1 plants; this damage was prevented by pretreatment with Gd³⁺. Our results suggest that copper transport into cytosol in root apex results in hydroxyl radical generation at the cytosolic side, with a consequent regulation of plasma membrane OH^?‐sensitive Ca²⁺ and K⁺ transport systems.     

SEB‐3, a CRF receptor‐like GPCR,regulates locomotor activity states,stress responses and ethanol tolerance in Caenorhabditis elegans

The CRF (corticotropin‐releasing factor) system is a key mediator of the stress response. Alterations in CRF signaling have been implicated in drug craving and ethanol consumption. The development of negative reinforcement via activation of brain stress systems has been proposed as a mechanism that contributes to alcohol dependence. Here, we isolated a gain‐of‐function allele of seb‐3, a CRF receptor‐like GPCR in Caenorhabditis elegans, providing an in vivo model of a constitutively activated stress system. We also characterized a loss‐of‐function allele of seb‐3 and showed that SEB‐3 positively regulates a stress response that leads to an enhanced active state of locomotion, behavioral arousal and tremor. SEB‐3 also contributed to acute tolerance to ethanol and to the development of tremor during ethanol withdrawal. Furthermore, we found that a specific CRF₁ receptor antagonist reduced acute functional tolerance to ethanol in mice. These findings demonstrate functional conservation of the CRF system in responses to stress and ethanol in vertebrates and invertebrates.     

Influence of Surface Chemistry on the Hygienic Status of Industrial Stainless Steel

Coupons of fourteen different stainless steels were investigated in terms of surface chemistry and ease of cleaning. Steel surfaces were exposed to Bacillus cereus spores in static saline solution for 2?h. Surfaces were rinsed and then covered with whole milk and allowed to dry. Surfaces were then cleaned in an experimental flow system that mimics an industrial application. After cleaning, remaining spores were released by sonication, spores cultured and colony forming units determined. Surfaces with higher levels of Fe in the outer surface of the passive film cleaned more easily. There was a relation between the polar component and ease of cleaning. The higher the polar component the more easily the surface cleaned. The cleaning mechanism involves dissolution of Fe enriched hydroxide films on the surface.     

An improved method using densitometry for evaluating severity of laser photocoagulation induced CNV

Laser photocoagulation induced choroidal neovascularization currently is the most effective model available for the study of this disease in terms of efficacy of new drugs and therapies. Previously, evaluating the extent of choroidal neovascularization using this model was time- consuming and required the use of experienced personnel. We describe a new method for simple and rapid evaluation of laser induced choroidal neovascularization using densitometry. Fluorescein angiograms of a laser photocoagulated rat eye were scanned into a computer. Densitometry software subsequently was used to calculate the severity of the laser lesions. The densitometry method proved effective for calculating the extent of laser induced choroidal neovascularization. In addition, this method was more rapid than visual evaluations and less likely to produce errors.