Acridine orange induces translocation of phosphatidylserine to red blood cell surface

Clustering of band-3 on red blood cell (RBC) surface has been assumed to catalyze RBC phagocytosis. In studying this subject, acridine orange (AO) has commonly been employed on the assumption that it specifically induces band-3 clustering. In the present study, we show that AO strongly induces translocation of phosphatidylserine (PS) to RBC surface. Because surface PS is well known to induce RBC intercellular interaction, these findings suggest that the use of AO as a specific inducer of band-3 clustering is questionable. It is possible that band-3 clustering and PS translocation are interdependent, and this interrelationship has yet to be explored. erythrocytes; adherence; acridine orange; band-3; phosphatidylserine     

T cells infiltrate the brain in murine and human transmissible spongiform encephalopathies

CD4 and CD8 T lymphocytes infiltrate the parenchyma of mouse brains several weeks after intracerebral, intraperitoneal, or oral inoculation with the Chandler strain of mouse scrapie, a pattern not seen with inoculation of prion protein knockout (PrP(-/-)) mice. Associated with this cellular infiltration are expression of MHC class I and II molecules and elevation in levels of the T-cell chemokines, especially macrophage inflammatory protein 1beta, IFN-gamma-inducible protein 10, and RANTES. T cells were also found in the central nervous system (CNS) in five of six patients with Creutzfeldt-Jakob disease. T cells harvested from brains and spleens of scrapie-infected mice were analyzed using a newly identified mouse PrP (mPrP) peptide bearing the canonical binding motifs to major histocompatibility complex (MHC) class I H-2(b) or H-2(d) molecules, appropriate MHC class I tetramers made to include these peptides, and CD4 and CD8 T cells stimulated with 15-mer overlapping peptides covering the whole mPrP. Minimal to modest K(b) tetramer binding of mPrP amino acids (aa) 2 to 9, aa 152 to 160, and aa 232 to 241 was observed, but such tetramer-binding lymphocytes as well as CD4 and CD8 lymphocytes incubated with the full repertoire of mPrP peptides failed to synthesize intracellular gamma interferon (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) cytokines and were unable to lyse PrP(-/-) embryo fibroblasts or macrophages coated with (51)Cr-labeled mPrP peptide. These results suggest that the expression of PrP(sc) in the CNS is associated with release of chemokines and, as shown previously, cytokines that attract and retain PrP-activated T cells and, quite likely, bystander activated T cells that have migrated from the periphery into the CNS. However, these CD4 and CD8 T cells are defective in such an effector function(s) as IFN-gamma and TNF-alpha expression or release or lytic activity.     

Function of herpes simplex virus type 1 gD mutants with different receptor-binding affinities in virus entry and fusion

We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(inverted Delta 34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(inverted Delta 34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its later function in virus entry. gD(inverted Delta 243t) showed WT binding affinity for both receptors, but virus containing this form of gD had a markedly reduced rate of entry, suggesting that gD(inverted Delta 243) is impaired in a postbinding step in the entry process. There was no correlation between gD mutant activity in fusion or virus entry and receptor-binding affinity. We conclude that gD functions in virus entry and cell fusion regardless of its receptor-binding kinetics and that as long as binding to a functional receptor occurs, entry will progress.     

Effect of enrichment on variation and results in the light/dark test

Several confounding factors may influence the outcome of an experiment and the extent of inter-individual variation. The aim of this study was to investigate if cage enrichment induces an effect on experimental mean values and on inter-individual variation in the light/dark paradigm using diazepam as the anxiolytic drug. The behaviour of 216 naive adult male mice of two different strains (BALB/c and C57BL/6) was studied. The animals were housed in groups of four in 'non-enriched', 'enriched' (nesting material) or 'super-enriched' (nest-box, nesting material, wooden gnawing stick and PVC tube) cages. After 5 weeks the animals were assigned to one of three treatments: control (no injection), sham (saline injection i.p.) or diazepam (1 mg/kg bw i.p.) and tested in the light/dark test for 5 min. Variation data were analysed using three different methods (mean absolute deviation, coefficient of variation and power analysis). The C57BL/6 mice scored higher than BALB/c mice in activity related measurements and showed a less 'emotional' behaviour profile in the pharmacological control situation of the light/dark test. In this study the anxiolytic effect of diazepam was clear in BALB/c mice but absent in C57BL/6 mice. Mice housed in enriched and super-enriched cages gained more weight than mice in non-enriched cages, although food intake was not affected. Generally, the strain of mouse had the greatest impact on both mean values and variation. However, there was no consistent increase for one particular strain. The choice of statistical method for analysing variation may influence the interpretation of within-group variability, but none of the methods showed any significant differences between standard and enriched conditions on variability in any of the parameters measured.     

Dynamics of the caring family

When several individuals simultaneously provide for offspring, as in families, the effort of any one individual will depend on the efforts of the other family members. This conflict of interest among family members is made more complicated by their relatedness because relatives share genetic interest to some degree. The conflict resolution will also be influenced by the differences in reproductive value between breeders and helpers. Here, we calculate evolutionarily stable provisioning efforts in families with up to two helpers. We explicitly consider that the behavioral choices are made in a life-history context, and we also consider how group sizes change dynamically; this affects, for example, average relatedness among group members. We assume two different scenarios: intact families in which the breeder is 100% monogamous and stepfamilies in which the breeder shifts mate between breeding events. The average relatedness among family members is allowed to evolve in concert with changes in provisioning effort. Our model shows that an individual's provisioning effort is not easy to predict from either its relatedness to the offspring or its reproductive value. Instead, it is necessary to consider the inclusive fitness effect of provisioning, which is determined by a combination of relatedness, reproductive value, and the reproductive value of the offspring.     

Metabolism of cyclic GMP in peritoneal macrophages of rat and guinea pig

The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca(2+)/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species.     

Modulation of the voltage-dependent anion-selective channel by cytoplasmic proteins from wild type and the channel depleted cells of Saccharomyces cerevisiae

It is well known that effective exchange of metabolites between mitochondria and the cytoplasm is essential for cell physiology. The key step of the exchange is transport across the mitochondrial outer membrane, which is supported by the voltage-dependent anion-selective channel (VDAC). Therefore, it is clear that the permeability of VDAC must be regulated to adjust its activity to the actual cell needs. VDAC-modulating activities, often referred to as the VDAC modulator, were identified in the intermembrane space of different organism mitochondria but the responsible protein(s) has not been identified as yet. Because the VDAC modulator was reported to act on VDAC of intact mitochondria when added to the cytoplasmic side it has been speculated that a similar modulating activity might be present in the cytoplasm. To check the speculation we used mitochondria of the yeast Saccharomyces cerevisiae as they constitute a perfect model to study VDAC modulation. The mitochondria contain only a single isoform of VDAC and it is possible to obtain viable mutants devoid of the channel (Deltapor1). Moreover, we have recently characterised a VDAC-modulating activity located in the intermembrane space of wild type and Deltapor1 S. cerevisiae mitochondria. Here, we report that the cytoplasm of wild type and Deltapor1 cells of S. cerevisiae contains a VDAC-modulating activity as measured in a reconstituted system and with intact mitochondria. Since quantitative differences were observed between the modulating fractions isolated from wild type and Deltapor1 cells when they were studied with intact wild type mitochondria as well as by protein electrophoresis it might be concluded that VDAC may influence the properties of the involved cytoplasmic proteins. Moreover, the VDAC-modulating activity in the cytoplasm differs distinctly from that reported for the mitochondrial intermembrane space. Nevertheless, both these activities may contribute efficiently to VDAC regulation. Thus, the identification of the proteins is very important.     

Selection of low-molecular-mass trypsin and chymotrypsin inhibitors based on the binding loop of CMTI-III using combinatorial chemistry methods

Using a combinatorial chemistry approach, a decapaptide library containing the N-terminal fragment of trypsin inhibitor CMTI-III was synthesized by the solid-phase method. The peptide library was screened for trypsin and chymotrypsin inhibitory activity applying the iterative method in solution. Two decapeptides were selected and resynthesized for each enzyme. The association equilibrium constants ((1.1+/-0.2)x10(8) and (7.3+/-1.6)x10(7)) determined for peptides with trypsin inhibitory activity indicate that they are 3-4-fold less active than the CMTI inhibitors. On the other hand, they are significantly more effective as compared with the starting sequence. Two peptides selected as chymotrypsin inhibitors displayed about 10 times higher activity (1.7+/-0.4)x10(7) and (1.1+/-0.2)x10(7), respectively) than those monosubstituted in position P(1) of the CMTI-III analogue. Considering low molecular weight of peptides selected and the lack of conformational constraints in their structures, the results are promising. They are good templates as starting sequences for further selection of small, peptidomimetic proteinase inhibitors.     

Preserved Tissue Allografts in Reconstructive Surgery

Results of treatment with three various kinds of allografts: lyophilized bone, deep frozen bone and cartilage preserved in physiological solution, all of them radiation-sterilized are presented. We believe that this presentation may be helpful in estimating the tissue bank's allografts and in establishing indications and contraindications in the application of allografts in surgery. The ‘indices of coincidence’ were compared in a group of 1014 patients after bone (lyophilized and radiation-sterilized) transplantation. It seems that such a variable as ‘rebuilding of graft’ may be of prognostic value in analysing the results of treatment in this group. The application of frozen and radiation-sterilized allogenic bone grafts for reconstructions is also described. An analysis of the results of treatment in 1125 patients reveals that the use of preserved bone reduces the extent and duration of surgery. Almost total substitution of grafts may be seen in 3–8 months after surgery. Allogenic, preserved cartilage is often used in facial reconstructions of face. Human costal cartilage, preserved in 0.9% NaCl and radiation-sterilized, was used for reconstruction. The patients were examined 24–190 months after surgery (in several clinical units) and results were collected in a special questionnaire by the team that performed surgery. In an analysed group of 437 patients after cartilage transplantation, 42.2% were operated because of posttraumatical changes, 29.0% because of congenital malformations and in 16.7% non-specific inflammations were the cause of reconstructive operations. Malformations were located mainly in the nose (59%), the ear concha (16.5%) and 10.9% were mandible. The results of treatment were compared with ages of patients, diagnosis and the locations of the changes. Very good results were achieved in 33.5% of the patients, and satisfactory in 41.8% of the patients. However, in 19.9% of the patients the result of treatment was unsatisfactory. Correlation between some clinical and biological characteristics and the result of treatment is under discussion. This revised version was published online in July 2006 with corrections to the Cover Date.