A role for galectin-3 in CD13-mediated homotypic aggregation of monocytes

We recently reported that anti-CD13 mAbs induce homotypic aggregation of monocytic cells. This phenomenon is signal transduction dependent and does not require CD13 aminopeptidase activity. Since CD13 is heavily glycosylated and a member of the galectin family (galectin-4) has been shown to associate with CD13 in the intestinal epithelium, we hypothesized that CD13-mediated aggregation might proceed through a carbohydrate-dependent mechanism involving galectin-3, the most highly expressed galectin on monocytes. We report here that lactose and anti-galectin-3 antibodies completely abrogate homotypic aggregation induced by anti-CD13 antibodies. Furthermore, galectin-3 co-immunoprecipitates with CD13 from resting U-937 cells and this association decreases during the aggregation process, a phenomenon that may have functional implications. Together, the results presented here point to a key role for galectin-3 in CD13-mediated homotypic aggregation of monocytic cells.     

The role of aquaporin 3 in the movement of water and cryoprotectants in mouse morulae

The permeability to water and cryoprotectants of the plasma membrane is crucial to the successful cryopreservation of embryos. Previously, we have shown in mouse morulae that water and glycerol move across the plasma membrane by facilitated diffusion, and we have suggested that aquaporin 3 plays an important role in their movement. In the present study, we clarify the contribution of aquaporin 3 to the movement of water and various cryoprotectants in mouse morulae by measuring the Arrhenius activation energies for permeability to cryoprotectants and water, through artificial expression of aquaporin 3 using Aqp3 cRNA in mouse oocytes, and by suppressing the expression of aquaporin 3 in morulae by injecting double-stranded RNA of Aqp3 at the one-cell zygote stage. The results show that aquaporin 3 plays an important role in the facilitated diffusion of water, glycerol, and ethylene glycol, but not of acetamide and dimethylsulfoxide. On the other hand, in a propylene glycol solution, aquaporin 3 in morulae transported neither propylene glycol nor water by facilitated diffusion, probably because of strong water-solute interactions. These results provide important information for understanding the permeability of the plasma membrane of the mouse embryo.     

Solution conformation of wild-type and mutant IgG3 and IgG4 immunoglobulins using crystallohydrodynamics: possible implications for complement activation

We have employed the recently described crystallohydrodynamic approach to compare the time-averaged domain orientation of human chimeric IgG3wt (wild-type) and IgG4wt as well as two hinge mutants of IgG3 and an IgG4S331P (mutation from serine to proline at position 331, EU numbering) mutant of IgG4. The approach involves combination of the known shape of the Fab and Fc regions from crystallography with hydrodynamic data for the Fab and Fc fragments and hydrodynamic and small angle x-ray scattering data for the intact IgG structures. In this way, ad hoc assumptions over hydration can be avoided and model degeneracy (uniqueness problems) can be minimized. The best fit model for the solution structure of IgG3wt demonstrated that the Fab regions are directed away from the plane of the Fc region and with a long extended hinge region in between. The best fit model of the IgG3m15 mutant with a short hinge (and enhanced complement activation activity) showed a more open, but asymmetric structure. The IgG3HM5 mutant devoid of a hinge region (and also devoid of complement-activation activity) could not be distinguished at the low-resolution level from the structure of the enhanced complement-activating mutant IgG3m15. The lack of inter-heavy-chain disulphide bond rather than a significantly different domain orientation may be the reason for the lack of complement-activating activity of the IgG3HM5 mutant. With IgG4, there are significant and interesting conformational differences between the wild-type IgG4, which shows a symmetric structure, and the IgG4S331P mutant, which shows a highly asymmetric structure. This structural difference may explain the ability of the IgG4S331P mutant to activate complement in stark contrast to the wild-type IgG4 molecule which is devoid of this activity.     

Identification and localization of calcium-dependent protease II inNeurospora crassa andUromyces appendiculatus

Summary The existence of Ca²⁺-dependent protease II in crude extracts ofNeurospora crassa andUromyces appendiculatus was demonstrated by immunoblotting using specific antibodies. In both extracts two immunoreacting bands were observed. The molecular mass of the major band inN. crassa corresponded to 37 kDa, while that inU. appendiculatus was 43 kDa, similar to that previously reported forAllomyces arbuscula. Immunofluorescence of the enzyme was predominantly localized in the apical regions of germlings and growing hyphae, suggesting a functional role for the enzyme in hyphal growth.     

The Use of Kinetic and Dynamic Data in Risk Assessment of Drugs

The risk assessment process for non-carcinogens must incorporate all available scientific information, including toxicokinetic and toxicodynamic data. The framework for exposure limit setting proposed by Renwick and the International Programme on Chemical Safety (IPCS) subdivides traditional 10X uncertainty factors (UFs) into separate partial-log default values based on kinetic and dynamic considerations and allows for incorporation of compound-specific data when available. In this investigation, an extensive literature search was conducted on nine pharmaceuticals in order to incorporate information on kinetics and dynamics to allow extrapolation across species and among susceptible humans. The drugs are diazepam, oxazepam, midazolam, buspirone, fluoxetine, venlafaxine, amlodipine, felodipine, and nifedipine. The composite factors were calculated using the highest ratio or the average ratio for appropriate parameters and default subfactor. For the drugs examined, most of the subfactors for kinetics and dynamics were less than the proposed values by Renwick and IPCS, and the composite factors were far less than 100. From this study, it was concluded that relevant compound-specific kinetic and dynamic data can reduce uncertainties associated with interspecies differences and interindividual variability.     

Light-induced degradation of cytochrome b559 during photoinhibition of the photosystem II reaction center.

The behaviour of cytochrome (cyt) b559 during acceptor- and donor-side photoinhibition has been investigated in oxygen-evolving and non-evolving photosystem II (PSII) membranes. Strong illumination at 20 degrees C under aerobiosis induced a strong decrease in the absorbance of the cyt b559 alpha-band in the two preparations. This absorbance decline was observed only in non-oxygen-evolving PSII samples when illumination was performed under aerobiosis but at 4 degrees C, or under anaerobiosis at 20 degrees C. These results suggest that acceptor-side photoinhibition induces the degradation of cyt b559 by a mechanism related to an enzymatic reaction mediated by singlet oxygen. Donor-side photoinhibition may induce, however, a non-enzymatic photocleavage of the protein.     

Isolation and Characterization of Alfalfa-Nodulating Rhizobia Present in Acidic Soils of Central Argentina and Uruguay

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809–1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N₂-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.     

Biomineralization of Carbonates by Marinococcus albus and Marinococcus halophilus Isolated from the Salar de Atacama (Chile)

We studied the precipitation of carbonates in 17 strains of moderately halophilic, Gram-positive cocci belonging to two species: Marinococcus halophilus and Marinococcus albus, isolated from the Salar de Atacama (Chile). They were cultivated in solid and liquid laboratory media for 42 days at salt concentrations (wt/vol) of 3%, 7.5%, 15%, and 20%. The bioliths precipitated were studied by X-ray diffraction and scanning electron microscopy. M. halophilus formed crystals at each of the salt concentrations, with a maximum number of strains capable of precipitating carbonates at 7.5% and 15% salt concentrations. M. albus did not precipitate at 20% and showed a maximum at 7.5%. This behavior is similar to that of other Gram-positive bacteria and differs from that found in Gram-negative bacteria. The bioliths precipitated were spherical, generally isolated, with a size of 10–100 μm, varying with salinity. They were of magnesium calcite (CO₃ Ca_1-x Mg_x) with Mg content increasing with increasing salinity and Mg/Ca molar ratio of the culture medium. These results demonstrate the active role played by M. halophilus and M. albus in the precipitation of carbonates. Received: 3 November 1998 / Accepted: 9 March 1999     

Regulation of c-Myc and Max in megakaryocytic and monocytic-macrophagic differentiation of K562 cells induced by protein kinase C modifiers: c-Myc is down-regulated but does not inhibit differentiation.

We have studied the regulation and role of c-Myc and Max in the differentiation pathways induced in K562 cells by 12-O-tetradecanoyl phorbol-13 acetate (TPA) and staurosporine, an activator and inhibitor, respectively, of protein kinase C (PKC). We found that staurosporine induced megakaryocytic differentiation, as revealed by the cellular ultrastructure, platelet formation, and DNA endoreduplication. In contrast, TPA induced a differentiated phenotype that more closely resembled that of the monocyte-macrophage lineage. c-myc expression was down-regulated in K562 differentiated by both TPA and staurosporine, whereas max expression did not change in either case. Although PKC enzymatic activity was low in cells terminally differentiated with TPA and staurosporine, inhibition of PKC activity by itself did not induce c-myc down-regulation. We conclude that the c-myc gene is switched off as a consequence of the differentiation process triggered by these drugs in a manner independent from PKC. Ectopic overexpression of c-Myc in K562 cells did not affect the monocytic-macrophagic and megakaryocytic differentiation, indicating that c-Myc suppression is not required for these processes in K562. Similarly, both differentiation pathways were not affected by Max overexpression or by concomitant overexpression of c-Myc and Max. This result is in contrast with the inhibition of erythroid differentiation of K562 exerted by c-Myc, suggesting divergent roles for c-Myc/Max, depending on the differentiation pathway.