Characterisation of the human APC1, the largest subunit of the anaphase-promoting complex

Accurate segregation of sister chromatids during mitosis is necessary to avoid the aneuploidy found in many cancers. The spindle checkpoint, which monitors the metaphase to anaphase transition, has been shown to be defective in cancers with chromosomal instability. This checkpoint regulates the anaphase-promoting complex or cyclosome (APC/C), a cell cycle ubiquitin ligase regulating among other things sister chromatid separation. We have previously investigated the mouse Apc1 protein (previously also called Tsg24), the largest subunit of the APC/C. We have now sequenced a full-length human APC1 cDNA, mapped its chromosomal location, and analysed its intron-exon boundaries. We have also investigated the RNA and protein expression of the Apc1 and other APC/C components in normal and cancer cells and the relative occurrence of expressed sequence tags (ESTs) representing APC subunits from different tissues. The different APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they perform their functions as part of a complex. A difference from this pattern is however seen for the APC6, which in some cases is more strongly expressed, suggesting a special function for this protein in certain tissues and cell types.     

Daunomycin accumulation and induction of programmed cell death in rat hair follicles

The anthracycline antibiotic daunomycin (DM) is useful for the treatment of leukemia but has side-effects such as alopecia. Using immunocytochemistry, we show that, after a single i.v. injection, DM accumulates in the nuclei of matrix cells and in the outer root sheath of hair follicles. DM-positive matrix cells are detectable up to 48 h after injection and exhibit a characteristic granular morphology, which is not observed in saline-injected controls. TUNEL-staining has revealed that DM injection induces programmed cell death (PCD) in rat hair follicles. Cells undergoing PCD are detectable as late as 5 days postinjection in both the matrix and outer root sheath. Newly developed double-staining has shown that some of the DM-positive matrix cell nuclei are also TUNEL-positive. Staining for activated caspase-3 has demonstrated immunopositive cells following DM administration both in the matrix and in the outer root sheath. Ultrastructural immunocytochemistry has shown the presence of DM-positive cells with two different types of morphology. About half of the immunopositive cells exhibit a morphology typical of classical apoptosis (PCD type 1), whereas the other half show signs of autophagic cell death (PCD type 2). Interestingly, little, if any, DM accumulation or apoptosis has been detected in the dermal hair papillae. This may have a bearing on potential regeneration of the hair follicles. Thus, DM accumulates in a characteristic pattern in hair follicles. This accumulation is associated with the induction of two morphologically distinct forms of PCD.     

Does UV-B influence biomass growth in lichens deficient in sun-screening pigments?

This study aimed to assess biomass growth as a response variable in lichens during short-term laboratory experiments. To do this, we studied the influence of UV-B and temperature on lichen performance including the synthesis of solar radiation screening cortical compounds. The pioneer lichen Xanthoria aureola from exposed sea cliffs and the old forest lichen Lobaria pulmonaria were cultivated for 15 days in the laboratory in a factorial experiments with temperature (12 and 21 °C) and UV-B (0, 0.1, 0.3 and 1.0 W m^?2) as treatments. Prior to the experiment, the cortical pigment parietin was non-destructively extracted from X. aureola, whereas the sampled shade-adapted thalli of L. pulmonaria lacked cortical melanic compounds. Therefore both lichens were deficient in cortical sun-screening compounds when the UV-B exposure started. At 12 °C, the relative growth rate was 7.2 ± 0.6 and 3.0 ± 0.8 mg g^?1 day^?1 in L. pulmonaria and X. aureola, respectively, reduced to 1.8 ± 0.5 and ?2.6 ± 0.9 mg g^?1 day^?1, at 21 °C. These figures showed that lichen growth is a useful response variable in short-term laboratory experiments. Growth was not influenced by UV-B alone in these pigment-deficient transplants, suggesting that UV-B had little adverse effects on either of the lichen bionts. The cortical sun screens (parietin and melanic compounds) were synthesized in the presence of UV-B, and increased statistically significantly with increasing UV-B at both cultivation temperatures. However, in X. aureola the synthesis was highest at the lowest temperature (12 °C). At 12 °C, changes in chlorophylls, F_v/F_m and NPQ during cultivation were consistent with a substantial level of acclimation to the growth chamber conditions for both species, whereas strong reductions in photosynthetic pigments, F_v/F_m and Ф_II at 21 °C indicated serious damage and chlorophyll degradation at high temperature. In conclusion, lichen growth and the synthesis of protective compounds are highly responsive lichen processes in short-term experiments.     

Distribution and hormonal regulation of membrane progesterone receptors β and γ in ciliated epithelial cells of mouse and human fallopian tubes

Background  

The controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma.     

Salivary cortisol differs with age and sex and shows inverse associations with WHR in Swedish women: a cross-sectional study

Background

The rising prevalence of type 2 diabetes underlines the importance of secondary strategies for the prevention of target organ damage. While access to diabetes education centers and diabetes intensification management has been shown to improve blood glucose control, these services are not available to all that require them, particularly in rural and northern areas. The provision of these services through the Home Care team is an advance that can overcome these barriers. Transfer of blood glucose data electronically from the home to the health care provider may improve diabetes management.

Methods and design

The study population will consist of patients with type 2 diabetes with uncontrolled A1c levels living on reserve in the Battlefords region of Saskatchewan, Canada. This pilot study will take place over three phases. In the first phase over three months the impact of the introduction of the Bluetooth enabled glucose monitor will be assessed. In the second phase over three months, the development of guidelines based treatment algorithms for diabetes intensification will be completed. In the third phase lasting 18 months, study subjects will have diabetes intensification according to the algorithms developed.

Discussion

The first phase will determine if the use of the Bluetooth enabled blood glucose devices which can transmit results electronically will lead to changes in A1c levels. It will also determine the feasibility of recruiting subjects to use this technology. The rest of the Diabetes Risk Evaluation and Management Tele-monitoring (DreamTel) study will determine if the delivery of a diabetes intensification management program by the Home Care team supported by the Bluetooth enabled glucose meters leads to improvements in diabetes management.

Trial Registration

Protocol NCT00325624     

Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8)

Auto-antibodies induced by cancer represent promising sensitive biomarkers and probes to identify immunotherapeutic targets without immunological tolerance. Surprisingly few epitopes for such auto-antibodies have been identified to date. Recently, a cancer-specific syngeneic murine monoclonal antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity of a chemoenzymatically produced Tn-glycopeptide derived from the putative murine podoplanin O-glycopeptide epitope. We found that the Tn O-glycopeptide was highly immunogenic in mice and produced a Tn-glycoform specific response with no reactivity against unglycosylated peptides or the O-glycopeptide with extended O-glycan (STn and T glycoforms). The immunodominant epitope was strictly dependent on the peptide sequence, required Tn at a specific single Thr residue (Thr⁷⁷), and antibodies to the epitope were not found in naive mice. We further tested a Tn O-glycopeptide library derived from human podoplanin by microarray analysis and demonstrated that the epitope was not conserved in man. We also tested human cancer sera for potential auto-antibodies to similar epitopes, but did not detect such antibodies to the Tn-library of podoplanin. The reagents and methods developed will be valuable for further studies of the nature and timing of induction of auto-antibodies to distinct O-glycopeptide epitopes induced by cancer. The results demonstrate that truncated O-glycopeptides constitute highly distinct antibody epitopes with great potential as targets for biomarkers and immunotherapeutics.     

Between-year variations in the development of crustacean zooplankton in the Norwegian subalpine lake, Øvre Heimdalsvatn

Data from the International Biological Programme (IBP) and subsequent studies have been re-analysed to test the two hypotheses which previously have been suggested concerning the zooplankton in the mountain lake, Øvre Heimdalsvatn: (1) the average temperature in June, more than other summer months, is affecting the growth rate and population densities of zooplankton in the lake, (2) the invasion of the European minnow (Phoxinus phoxinus) has caused changes in the zooplankton community. The analyses have demonstrated that the June temperature strongly affects the growth rate of all the zooplankton species, but that there is no relationship with the population maxima. The species composition in the crustacean zooplankton has not changed between 1969 and 1999, and any direct impact of the minnows on the zooplankton community could not be detected. Indirectly, the minnows may have reduced the density of invertebrate predators, and thus caused an increase in juvenile survival and increased summer maximum density of Bosmina longispina. The variation in density of the copepod, Cyclops scutifer, was correlated with the density of Heterocope saliens, most likely the result of predator–prey interactions.     

Design of an Optimized Scaffold for Affibody Molecules

Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z_HER2:342. Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to + 12 °C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z_HER2:2891-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K_D, of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 °C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor α, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor β) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.     

Effects of aging and gender on the spatial organization of nuclei in single human skeletal muscle cells

The skeletal muscle fibre is a syncitium where each myonucleus regulates the gene products in a finite volume of the cytoplasm, i.e., the myonuclear domain (MND). We analysed aging‐ and gender‐related effects on myonuclei organization and the MND size in single muscle fibres from six young (21–31 years) and nine old men (72–96 years), and from six young (24–32 years) and nine old women (65–96 years), using a novel image analysis algorithm applied to confocal images. Muscle fibres were classified according to myosin heavy chain (MyHC) isoform expression. Our image analysis algorithm was effective in determining the spatial organization of myonuclei and the distribution of individual MNDs along the single fibre segments. Significant linear relations were observed between MND size and fibre size, irrespective age, gender and MyHC isoform expression. The spatial organization of individual myonuclei, calculated as the distribution of nearest neighbour distances in 3D, and MND size were affected in old age, but changes were dependent on MyHC isoform expression. In type I muscle fibres, average NN‐values were lower and showed an increased variability in old age, reflecting an aggregation of myonuclei in old age. Average MND size did not change in old age, but there was an increased MND size variability. In type IIa fibres, average NN‐values and MND sizes were lower in old age, reflecting the smaller size of these muscle fibres in old age. It is suggested that these changes have a significant impact on protein synthesis and degradation during the aging process.