EPR properties of mixed-valent μ-oxo and μ-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K

 Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S=1/2 ground state with small g-anisotropy as characterized by the uniaxial component (g _z –g _av /2<0.03) observable at temperatures as high as ∼100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (g _z –g _av /2>0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins. Received: 27 June 1998 / Accepted: 25 February 1999     

Mesothelioma Tumor Cells Modulate Dendritic Cell Lipid Content,Phenotype and Function

Dendritic cells (DCs) play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs) were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay), upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4⁺CD8α^- DCs, CD4^-CD8α^- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8⁺ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.     

Liver Accumulation of Plasmodium chabaudi-Infected Red Blood Cells and Modulation of Regulatory T Cell and Dendritic Cell Responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (Pc-iRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.     

Estimates of the Prevalence of Obesity in Canadian Children

Childhood obesity is becoming a topic of great concern due to the rising prevalence of this condition in North America. Studies conducted in the United States have indicated that the prevalence of obesity has increased dramatically over the past few decades. The purpose of this study was to estimate the prevalence of obesity in Canadian children between the ages of 5 and 12 years by examining data from two national and two regional surveys. The 85th percentiles of each of four anthropometric indices derived from large normative populations were used as diagnostic criteria for obesity. As expected, the resulting prevalences varied according to the criteria used. A significant increase in childhood obesity between the 1981 to 1988 national surveys was observed when the three indices which used skinfolds were applied. Weight-for-height percentiles did not indicate an increase in obesity in these samples. Regional samples showed a less than expected prevalence of obesity among the middle-class children and a higher than expected rate among the inner city boys. It can be concluded that there is a need for a defined criteria for identifying obesity in children in order to avoid confusion resulting from the wide variation in estimates of prevalence resulting from different standards and measurements. Using adiposity-based criteria for obesity it was clearly evident that the prevalence of obesity has increased in Canadian children.     

Regulation of Dipeptidyl Aminopeptidase I and Angiotensin Converting Enzyme Activities in Cultured Murine Brain Cells by Cortisol and Thyroid Hormone

Cultures of dissociated brain cells from 15-day-old fetal mice were grown in the presence and absence of 20 or 50 nM triiodothyronine (T3), 30 or 300 nM cortisol, and 30 nM cortisol plus 50 nM T3 added to chemically defined media or in media supplemented with 15% serum from control and hypothyroid calves. The specific activities of five lysosomal enzymes--N-acetyl galactosaminidase, beta-glucuronidase, beta-galactosidase, cathepsin B, and dipeptidyl aminopeptidase I (DAP-I)--were higher in cells grown in calf serum than in cells grown in defined media. Of these enzymes, only DAP-I was elevated in activity when the cells were grown in hypothyroid calf serum instead of control calf serum. Elevation of DAP-I activity was reversed by addition of 20 nM T3 to hypothyroid calf serum. The enzymatic properties of DAP-I were similar whether the cells were grown in control or hypothyroid calf serum and were similar to those reported for human fibroblasts and the purified enzyme. When the cells were grown in defined media, cortisol decreased the activities of all lysosomal enzymes, with 300 nM cortisol being more effective than 30 nM cortisol. Addition of 50 nM T3 to 30 nM cortisol decreased DAP-I activity more than 30 nM cortisol alone, but 50 nM T3 alone in defined media did not alter DAP-I levels. The reduction of DAP-I activity in these cells by T3 required cortisol, unidentified components in serum, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

The alpha-A-crystallin and cystathionine beta-synthase genes are physically very closely linked in proximal mouse chromosome 17

Murine genes homologous to those contributing to the Down syndrome (DS) phenotype in man are currently of interest because of their potential for providing animal models for the study of specific DS symptoms. Most of the genes mapping to human chromosome 21q22, where the DS genes are concentrated, are related to sequences located on mouse chromosome 16. Others, however, are known to map to mouse chromosome 10, and two genes, cystathionine beta-synthase (Cbs) and alpha-A-crystallin (Crya-1), have been localized to the proximal portion of mouse chromosome 17. In this paper, we show that the two genes mapping to human chromosome 21q22 and mouse chromosome 17 are very tightly linked in mouse, being separated by at least 70 kb, but not more than 130 kb. The very close physical linkage of mouse Cbs and Crya-1, combined with data that localize homologs of the closely flanking markers H2k and Pim-1 to human chromosome 6, suggests that the human 21q22/mouse chromosome 17 conserved segment is of a very limited total physical size and is likely to contain a relatively small number of genes.     

The HOX-5 and surfeit gene clusters are linked in the proximal portion of mouse chromosome 2

Using an interspecies backcross, we have mapped the HOX-5 and surfeit (surf) gene clusters within the proximal portion of mouse chromosome 2. While the HOX-5 cluster of homeobox-containing genes has been localized to chromosome 2, bands C3-E1, by in situ hybridization, its more precise position relative to the genes and cloned markers of chromosome 2 was not known. Surfeit, a tight cluster of at least six highly conserved “housekeeping” genes, has not been previously mapped in mouse, but has been localized to human chromosome 9q, a region of the human genome with strong homology to proximal mouse chromosome 2. The data presented here place HOX-5 in the vicinity of the closely linked set of developmental mutations rachiterata, lethargic, and fidget and place surf close to the proto-oncogene Abl, near the centromere of chromosome 2.     

Plant wound compounds from oviposition scars used in host discrimination by a stem-galling sawfly

The stem-galling sawfly Euura lasiolepisuses one or more plant wound compounds resulting from oviposition scars as cues in host discrimination (avoiding sites occupied by conspecifics). Four experiments were conducted to test hypotheses about how Euurapartitions resources. Experiment 1 demonstrated that Euuraavoids ovipositing on nodes with scars from previous ovipositions. Experiment 2 showed no evidence that the sawfly uses oviposition-deterring pheromones and indicated there is a time lag following oviposition before the oviposition scar becomes a deterrent. Experiment 3 showed that sawflies avoid artificially formed scars, demonstrating that a plant cue alone can lead to host discrimination. Experiment 4 showed that visual or tactile cues are not necessary for host discrimination and indicated that a plant wound compound functions as an oviposition deterrent. Both experimental results and field surveys showed that Euuraoviposition scars were more uniformly distributed than expected if sawflies were ignoring previous ovipositions.     

Long-range walking techniques in positional cloning strategies

Conclusion The past several years have seen an explosive growth in our understanding of the organization and structure of mammalian genomes, and refinements of existing techniques for genetic analysis, physical mapping, and large-fragment cloning techniques may well be enough to continue the momentum of that explosion for some time to come. Although refinement of existing techniques will certainly be necessary, the development of new and better cloning techniques may, perhaps, no longer be our most urgent need. The most important challenge that we face at present may in fact be that of finding efficient ways to share existing resources and information rapidly and equitably throughout the scientific community so that progress can continue unimpeded, and to catalog, correlate, and interpret the wealth of new data that is so rapidly accumulating.New strategies aimed at whole-genome mapping (Coulson et al. 1986, 1988; Michiels et al. 1987; Brenner and Livak 1989; Carrano et al. 1989; Lehrach et al. 1991) and sequencing (Church and Keifer-Higgins 1988; Bains and Smith 1988; Drmanac et al. 1989; Strzoska et al. 1991) may someday make the current method of long-range walking and physical mapping nearly passe. For example, since most of the relatively small nematode genome is now stored as ordered sets of cosmid and YAC clones (Coulson et al. 1986, 1988), a walk between a mapped marker and an uncloned gene can be accomplished rapidly, through a request for the appropriate series of clones from the ordered library. Vigorous drives by many laboratories to produce ordered clone libraries for murine and human chromosomes (Lehrach et al. 1991) may transform the process of cloning mammalian genes into a relatively trivial matter within the foreseeable future. The remarkable number of positional-cloning successes that have been reported in recent years may indicate that most of the best-defined, simply inherited mouse mutations and human hereditary disorders will have already been cloned by that time. When that is accomplished, the true challenging task will just begin: we must learn to decipher the complex biological programs encoded by our large and ever-growing storehouse of cloned, mapped and sequenced genes, before we can begin to understand what might be held in the vast silent mass of mammalian genomes. Offprint requests to: L. Stubbs