Methylation of CpG dinucleotides in the lacI gene of the Big Blue® transgenic mouse

Cytosine residues at CpG dinucleotides can be methylated by endogenous methyltransferases in mammalian cells. The resulting 5-methylcytosine base may undergo spontaneous deamination to form thymine causing G/C to A/T transition mutations. Methylated CpGs also can form preferential targets for environmental mutagens and carcinogens. The Big Blue® transgenic mouse has been used to investigate tissue and organ specificity of mutations and to deduce mutational mechanisms in a mammal in vivo. The transgenic mouse contains approximately 40 concatenated lambda-like shuttle vectors, each of which contains one copy of an Escherichia coli lacI gene as a mutational target. lacI mutations in lambda transgenic mice are characterized by a high frequency of spontaneous mutations targeted to CpG dinucleotides suggesting an important contribution from methylation-mediated events. To study the methylation status of CpGs in the lacI gene, we have mapped the distribution of 5-methylcytosines along the DNA-binding domain and flanking sequences of the lacI gene of transgenic mice. We analyzed genomic DNA from various tissues including thymus, liver, testis, and DNA derived from two thymic lymphomas. The mouse genomic DNAs and methylated and unmethylated control DNAs were chemically cleaved, then the positions of 5-methylcytosines were mapped by ligation-mediated PCR which can be used to distinguish methylated from unmethylated cytosines. Our data show that most CpG dinucleotides in the DNA binding domain of the lacI gene are methylated to a high extent (>98%) in all tissues tested; only a few sites are partially (70–90%) methylated. We conclude that tissue-specific methylation is unlikely to contribute significantly to tissue-specific mutational patterns, and that the occurrence of common mutation sites at specific CpGs in the lacI gene is not related to selective methylation of only these sequences. The data confirm previous suggestions that the high frequency of CpG mutations in lacI transgenes is related to the presence of 5-methylcytosine bases.     

Time to Integrate to Nest Test Evaluation in a Mouse DSS-Colitis Model

Severity assessment in laboratory animals is an important issue regarding the implementation of the 3R concept into biomedical research and pivotal in current EU regulations. In mouse models of inflammatory bowel disease severity assessment is usually undertaken by clinical scoring, especially by monitoring reduction of body weight. This requires daily observance and handling of each mouse, which is time consuming, stressful for the animal and necessitates an experienced observer. The time to integrate to nest test (TINT) is an easily applicable test detecting disturbed welfare by measuring the time interval mice need to integrate nesting material to an existing nest. Here, TINT was utilized to assess severity in a mouse DSS-colitis model. TINT results depended on the group size of mice maintained per cage with most consistent time intervals measured when co-housing 4 to 5 mice. Colitis was induced with 1% or 1.5% DSS in group-housed WT and Cd14-deficient mice. Higher clinical scores and loss of body weight were detected in 1.5% compared to 1% DSS treated mice. TINT time intervals showed no dose dependent differences. However, increased clinical scores, body weight reductions, and increased TINT time intervals were detected in Cd14 ^-/- compared to WT mice revealing mouse strain related differences. Therefore, TINT is an easily applicable method for severity assessment in a mouse colitis model detecting CD14 related differences, but not dose dependent differences. As TINT revealed most consistent results in group-housed mice, we recommend utilization as an additional method substituting clinical monitoring of the individual mouse.     

Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers

Introduction  

Development of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes.     

The rate of oxygen utilization by cells

The discovery of oxygen is considered by some to be the most important scientific discovery of all time—from both physical-chemical/astrophysics and biology/evolution viewpoints. One of the major developments during evolution is the ability to capture dioxygen in the environment and deliver it to each cell in the multicellular, complex mammalian body—on demand, i.e., just in time. Humans use oxygen to extract approximately 2550 calories (10.4 MJ) from food to meet daily energy requirements. This combustion requires about 22 mol of dioxygen per day, or 2.5 × 10^− 4 mol s^− 1. This is an average rate of oxygen utilization of 2.5 × 10^− 18 mol cell^− 1 s^− 1, i.e., 2.5 amol cell^− 1 s^− 1. Cells have a wide range of oxygen utilization, depending on cell type, function, and biological status. Measured rates of oxygen utilization by mammalian cells in culture range from < 1 to > 350 amol cell^− 1 s^− 1. There is a loose positive linear correlation of the rate of oxygen consumption by mammalian cells in culture with cell volume and cell protein. The use of oxygen by cells and tissues is an essential aspect of the basic redox biology of cells and tissues. This type of quantitative information is fundamental to investigations in quantitative redox biology, especially redox systems biology.     

Cloning, expression and characterization of the murine orthologue of SBF2, the gene mutated in Charcot-Marie-Tooth disease type 4B2

Autosomal recessive hereditary motor and sensory neuropathy (HMSN) or Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous disorder of the peripheral nervous system. The clinical picture includes progressive distal weakness and atrophy, foot deformities, and distal sensory loss. For autosomal recessive CMT type 4B2 one locus was mapped to chromosome 11p15. Recently, mutations in SET binding factor 2 (SBF2), were identified as cause of CMT4B2. SBF2 is a member of the pseudo-phosphatase branch of myotubularins and all disease-associated mutations known to date lead to shortened or truncated proteins, also implicating loss-of-function. Here, we describe the molecular cloning and the expression pattern of Sbf2. The mRNA spans around 8 kb, and the protein shares high amino acid identity compared to the human protein suggesting a conserved function. Sbf2 is encoded by 40 exons on murine chromosome 7. In situ hybridization, Northern blots and RT-analysis revealed a very broad pattern of Sbf2 expression. Overexpressed epitope tagged Sbf2 showed cytoplasmic distribution. Taken together, this study provides information about the mRNA expression and subcellular localization of Sbf2 and as such helps in further understanding its function in development and disease.     

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells

As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.     

Short term voluntary overfeeding disrupts brain insulin control of adipose tissue lipolysis

Insulin controls fatty acid (FA) release from white adipose tissue (WAT) through direct effects on adipocytes and indirectly through hypothalamic signaling by reducing sympathetic nervous system outflow to WAT. Uncontrolled FA release from WAT promotes lipotoxicity, which is characterized by inflammation and insulin resistance that leads to and worsens type 2 diabetes. Here we tested whether early diet-induced insulin resistance impairs the ability of hypothalamic insulin to regulate WAT lipolysis and thus contributes to adipose tissue dysfunction. To this end we fed male Sprague-Dawley rats a 10% lard diet (high fat diet (HFD)) for 3 consecutive days, which is known to induce systemic insulin resistance. Rats were studied by euglycemic pancreatic clamps and concomitant infusion of either insulin or vehicle into the mediobasal hypothalamus. Short term HFD feeding led to a 37% increase in caloric intake and elevated base-line free FAs and insulin levels compared with rats fed regular chow. Overfeeding did not impair insulin signaling in WAT, but it abolished the ability of mediobasal hypothalamus insulin to suppress WAT lipolysis and hepatic glucose production as assessed by glycerol and glucose flux. HFD feeding also increased hypothalamic levels of the endocannabinoid 2-arachidonoylglycerol after only 3 days. In summary, overfeeding impairs hypothalamic insulin action, which may contribute to unrestrained lipolysis seen in human obesity and type 2 diabetes.     

Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis

The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme.