Self-assembly of Pseudomonas fluorescens lipase into bimolecular aggregates dramatically affects functional properties

It has been found that lipase from Pseudomonas fluorescens (PFL) is able to aggregate into bimolecular structures (MW around 66 kD) even at moderate enzyme concentrations. At very low enzyme concentrations and in the presence of detergents, the same enzyme displayed a unimolecular structure with a molecular weight of 33 kD. Both enzyme structures displayed different functional properties. First, the bimolecular structure was much more stable than the unimolecular species (the bimolecular structure maintained over 80% of initial activity after 72 hours at 45 degrees C, while the unimolecular structure retained only around 30% of initial activity after 4 hours of incubation under the same experimental conditions); and the bimolecular form presented a higher optimal T. Second, the unimolecular form showed a much lower K(M) for ethyl butyrate than the bimolecular form. Third, the interfacial activation in biphasic substrate-aqueous milieu was higher for the bimolecular form. Fourth, the unimolecular structure was less active but much more enantioselective than the unimolecular species in the model reaction used. It is proposed that the bimolecular aggregates of PFL might be formed by two open lipase molecules (mutual interfacial activation), in intimate contact, and that the bimolecular form represents an example of "pseudo-quaternary" structure.     

Evidence for glycosylphosphatidylinositol (GPI)-anchored eosinophil-derived neurotoxin (EDN) on human granulocytes

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST–SLAP-130–SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells.     

Differential activities of the SoxR protein of Escherichia coli: SoxS is not required for gene activation under iron deprivation

When Escherichia coli cells are under superoxide stress, proteins SoxR and SoxS, acting sequentially, control the expression of a set of repair and defense genes. One of these genes, fumC, encoding fumarase C, was reported to be also activated by iron deprivation in a soxRS-dependent manner. However, the same condition failed to induce the expression of a soxS'::lacZ fusion. The expression of acnA (aconitase A) is also activated by SoxR alone when under iron deprivation, but not of sodA (Mn-superoxide-dismutase). SoxR completely inhibited the migration of a DNA fragment containing the promoter region of fumC, in gel-shift experiments. SoxR might bind to a different region than SoxS within the fumC promoter, or an unknown intermediate other than SoxS might be acting. It is possible that the regulatory role of SoxR is more complex than previously considered.     

MICA-A5.1 allele is associated with atypical forms of celiac disease in HLA-DQ2-negative patients

We selected 38 consecutive celiac disease (CD) patients (from a group of 316 consecutive CD patients) and 91 healthy blood donors, all of whom were HLA-DQ2 (DQA1*0501/DQB1*0201) negative, and investigated the presence of the classically associated alleles HLA-DQ8 and HLA-DRB4. We also studied the distribution of MICA transmembrane alleles in the two clinical forms of the disease. For this reason, these 38 DQ2-negative patients were subdivided into two groups: 18 typical CD patients and 20 atypical CD patients. No differences were found in the distribution of the DRB4 allele between DQ2-negative patients and controls. The HLA-DQ8 heterodimer (DQA1*03xx/DQB1*0302) was increased in CD patients (29%) compared with controls (10%), but no statistical differences were found. No differences were observed in the frequency of these alleles between either group of CD DQ2-negative patients. MICA-A5.1 was increased in atypical CD patients when compared with the typical forms of disease ( P(c)=0.03) and with healthy controls (P(c)=0.002). No other MICA allele was found to be significantly increased in the groups under study. The presence of MICA-A5.1 in atypical CD DQ2-negative patients may indicate a possible role of this allele in the development of CD.     

Conflict and post-conflict behavior in a small group of chimpanzees

Chimpanzee research plays a central role in the discussions of conflict negotiation. Reconciliation, or the attraction and affiliation of former opponents following conflict, has been proposed as a central element of conflict negotiation in chimpanzees and various other taxa. In an attempt to expand the database of chimpanzee conflict resolution, conflict and post-conflict behavior were recorded for a small group of socially housed chimpanzees at the Chimpanzee and Human Communication Institute, at Central Washington University. Data were collected over six 6-week periods between 1997 and 2000, for a total of 840 hours of observation, resulting in a substantial post-conflict (PC) and matched control (MC) data set. The data demonstrate this group’s tendencies to maintain visual contact and closer proximity after conflicts. Dyadic corrected conciliatory tendencies ranged between 0 – 37.5% and averaged 17.25% across all dyads. Individual corrected conciliatory tendencies ranged between 5.8 and 32%. The results of this study combined with recent publications on captive and free-ranging chimpanzee post-conflict behavior suggest that variation in post-conflict behavior may be important to our understanding of chimpanzee conflict negotiation, and may also have implications for the design and management of captive chimpanzee enclosures and social groups, respectively.     

Enzymatic acylation of di- and trisaccharides with fatty acids: choosing the appropriate enzyme,support and solvent

Enzymatic synthesis of fatty acid esters of di- and trisaccharides is limited by the fact that most biological catalysts are inactivated by the polar solvents (e.g. dimethylsulfoxide, dimethylformamide) where these carbohydrates are soluble. This article reviews the methodologies developed to overcome this limitation, namely those involving control over the reaction medium, the enzyme and the support. We have proposed the use of mixtures of miscible solvents (e.g. dimethylsulfoxide and 2-methyl-2-butanol) as a general strategy to acylate enzymatically hydrophilic substrates. We observed that decreasing the hydrophobicity of the medium (i.e. lowering the percentage of DMSO) the molar ratio sucrose diesters versus sucrose monoesters can be substantially enhanced. The different regioselectivity exhibited by several lipases and proteases makes feasible to synthesise different positional isomers, whose properties may vary considerably. In particular, the lipase from Thermomyces lanuginosus displays a notable selectivity for only one hydroxyl group in the acylation of sucrose, maltose, leucrose and maltotriose, compared with lipase from Candida antarctica. We have examined three immobilisation methods (adsorption on polypropylene, covalent coupling to Eupergit C, and silica-granulation) for sucrose acylation catalysed by T. lanuginosus lipase. The morphology of the support affected significantly the reaction rate and/or the selectivity of the process.     

Sexual exhaustion in White New Zealand male rabbits of different ages

Sexual exhaustion was studied in hybrid White New Zealand rabbits of different ages, for this purpose six young rabbits from 6 to 12 months of age, and six adult rabbits of 14-20 months of age were selected. Sexually receptive females were taken to the male's cage, for a period of 4 min, if copulation was not performed, the observation was then considered finished. If the male mounted within this period of time, the mounted female was immediately replaced by another female and 4 min of exposure time to the male were reinitiated, and repetitions were conducted until the male that was being studied refused to mount a new female at which time the male was considered to be sexually exhausted. Young rabbits mounted and ejaculated 9-10 times before sexual exhaustion. Adult rabbits showed a fluctuation of between 6 and 8 mounts per ejaculations before refusing another mount. The statistical analysis with a Mann-Whitney U-test, showed that the Rank sum for group A was 57, while for group B was 21. The U-value was 0.0 and the adjusted Z -2.9943. A significant difference was observed between groups with a value of P=0.00275. In the present study it was demonstrated that there is an influence of age on sexual behavior of rabbits.     

On-line analysis of gap junctions reveals more efficient electrical than dye coupling between islet cells

Pancreatic beta-cells constitute a well-communicating multicellular network that permits a coordinated and synchronized signal transmission within the islet of Langerhans that is necessary for proper insulin release. Gap junctions are the molecular keys that mediate functional cellular connections, which are responsible for electrical and metabolic coupling in the majority of cell types. Although the role of gap junctions in beta-cell electrical coupling is well documented, metabolic communication is still a matter of discussion. Here, we have addressed this issue by use of a fluorescence recovery after photobleaching (FRAP) approach. This technique has been validated as a reliable and noninvasive approach to monitor functional gap junctions in real time. We show that control pancreatic islet cells did not exchange a gap junction-permeant molecule in either clustered cells or intact islets of Langerhans under conditions that allowed cell-to-cell exchange of current-carrying ions. Conversely, we have detected that the same probe was extensively transferred between islet cells of transgenic mice expressing connexin 32 (Cx32) that have enhanced junctional coupling properties. The results indicate that the electrical coupling of native islet cells is more extensive than dye communication. Dye-coupling domains in islet cells appear more restricted than previously inferred with other methods.     

Inducible nitric oxide synthase inhibitors suppress airway inflammation in mice through down-regulation of chemokine expression

Growing evidence demonstrates that inducible NO synthase (iNOS) is induced in the airways of asthmatic patients. However, the precise role of NO in the lung inflammation is unknown. This study investigated the effect of both selective and nonselective iNOS inhibitors in an allergen-driven murine lung inflammation model. OVA challenge resulted in an accumulation of eosinophils and neutrophils in the airways. Expression of iNOS immunostaining in lung sections together with an increase in calcium-independent NOS activity in lung homogenates was also observed after OVA challenge. Treatment with iNOS inhibitors from the day of challenge to the day of sacrifice resulted in an inhibition of the inflammatory cell influx together with a down-regulation of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 production. In contrast, eosinophilic and neutrophilic inhibition was not observed with treatment during the sensitization. Both treatments induced an increased production of Th2-type cytokines (IL-4 and IL-5) with a concomitant decrease in production of Th1-type cytokine (IFN-gamma). In vitro exposure of primary cultures of murine lung fibroblasts to a NO donor, hydroxylamine, induced a dose-dependent release of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1. Our results suggest that lung inflammation after allergen challenge in mice is partially dependent on NO produced mainly by iNOS. NO appears to increase lung chemokine expression and, thereby, to facilitate influx of inflammatory cells into the airways.