Determination of thiopurine S-methyltransferase (TPMT) activity by comparing various normalization factors: reference values for Estonian population using HPLC-UV assay

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) is the key enzyme in the metabolism of thiopurine drugs. Determination of TPMT activity has been used for the individualization of thiopurine dose. We developed HPLC-UV assay for the determination of TPMT activity in human erythrocytes using 6-mercaptopurine as a substrate. Various extraction and chromatographic conditions were compared. In-house developed extraction with acetonitrile provided the lowest limit of quantification. TPMT activity was determined in 99 previously genotyped healthy Estonians. TPMT activity was expressed as the formation of 6-methylmercaptopurine ng/ml/h and normalized either to haemoglobin, haematocrit, erythrocyte count or protein content. The receiver-operating characteristic curve analysis revealed similar accuracy values for TPMT activity in predicting heterozygous and wild type individuals for each method of calculation. In healthy Estonians, TPMT activity varied from 21.5 to 129.6 ng/ml/h. For heterozygous individuals (n = 18), TPMT activity was 48.1 +/- 11.7 ng/ml/h. Wild type individuals (n = 81) revealed significantly higher TPMT activity 79.3 +/- 20.7 ng/ml/h (P < 0.001). This sensitive HPLC assay for quantitative determination of TPMT activity could easily be used in clinical settings. Under constant experimental conditions for haemolysate preparation no normalization is required.     

Metabolic control analysis of cellular respiration in situ in intraoperational samples of human breast cancer

The aim of this study was to analyze quantitatively cellular respiration in intraoperational tissue samples taken from human breast cancer (BC) patients. We used oxygraphy and the permeabilized cell techniques in combination with Metabolic Control Analysis (MCA) to measure a corresponding flux control coefficient (FCC). The activity of all components of ATP synthasome, and respiratory chain complexes was found to be significantly increased in human BC cells in situ as compared to the adjacent control tissue. FCC(s) were determined upon direct activation of respiration with exogenously-added ADP and by titrating the complexes with their specific inhibitors to stepwise decrease their activity. MCA showed very high sensitivity of all complexes and carriers studied in human BC cells to inhibition as compared to mitochondria in normal oxidative tissues. The sum of FCC(s) for all ATP synthasome and respiratory chain components was found to be around 4, and the value exceeded significantly that for normal tissue (close to 1). In BC cells, the key sites of the regulation of respiration are Complex IV (FCC?=?0.74), ATP synthase (FCC?=?0.61), and phosphate carrier (FCC?=?0.60); these FCC(s) exceed considerably (~10-fold) those for normal oxidative tissues. In human BC cells, the outer mitochondrial membrane is characterized by an increased permeability towards adenine nucleotides, the mean value of the apparent K(m) for ADP being equal to 114.8?±?13.6?μM. Our data support the two-compartment hypothesis of tumor metabolism, the high sum of FCC(s) showing structural and functional organization of mitochondrial respiratory chain and ATP synthasome as supercomplexes in human BC.     

Coccidian infection causes oxidative damage in greenfinches

The main tenet of immunoecology is that individual variation in immune responsiveness is caused by the costs of immune responses to the hosts. Oxidative damage resulting from the excessive production of reactive oxygen species during immune response is hypothesized to form one of such costs. We tested this hypothesis in experimental coccidian infection model in greenfinches Carduelis chloris. Administration of isosporan coccidians to experimental birds did not affect indices of antioxidant protection (TAC and OXY), plasma triglyceride and carotenoid levels or body mass, indicating that pathological consequences of infection were generally mild. Infected birds had on average 8% higher levels of plasma malondialdehyde (MDA, a toxic end-product of lipid peroxidation) than un-infected birds. The birds that had highest MDA levels subsequent to experimental infection experienced the highest decrease in infection intensity. This observation is consistent with the idea that oxidative stress is a causative agent in the control of coccidiosis and supports the concept of oxidative costs of immune responses and parasite resistance. The finding that oxidative damage accompanies even the mild infection with a common parasite highlights the relevance of oxidative stress biology for the immunoecological research.     

Structure of Mitochondria and Activity of Their Respiratory Chain in Successive Generations of Yeast Cells Exposed to He-Ne Laser Light

The data on the aftereffect of He-Ne laser light (λ = 632.8 nm) on mitochondria of yeasts in late log phase were reviewed. The quantitative analysis of the ultrathin cell sections demonstrated a nonuniform thickness of the giant branched mitochondria typical for budding yeasts. Exposure to a dose of 460 J/m² (accelerating cell proliferation and activating respiratory chain enzymes, cytochrome c oxidase and NADH dehydrogenase), changed the macrostructure of the giant mitochondria—much of the narrow regions of the mitochondrial tube with profiles ≤0.06 μm² were expanded (while no signs of organelle damage were observed). Such mitochondria are characterized by increased relative surface area of the cristae, which can be due to the activation of their respiration and ATP synthesis. The number of associations between mitochondria and endoplasmic reticulum increased in irradiated cells in early log phase, which reflects the increased capacity of mitochondria to uptake Ca²⁺. Altered giant mitochondria configuration can increase the efficiency of both energy transfer and Ca²⁺ propagation through the cytoplasm. __________ Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 6, 2005, pp. 672–683. Original Russian Text Copyright ? 2005 by Manteifel, Karu.     

Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.     

Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH₂ groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks.     

Polyamine depletion with two different polyamine analogues causes DNA damage in human breast cancer cell lines

It is well known that the positively charged polyamines have a DNA-stabilizing function and that polyamine depletion alters chromatin function. We have previously shown that polyamine depletion causes an S phase prolongation, and others have shown that there is an accumulation of Okazaki-like fragments in polyamine-depleted cells. In the present study, we have used the comet assay to investigate polyamine depletion-induced DNA strand breaks. Three breast cancer cell lines and one normal-like breast cell line were treated with the polyamine analogue N(1),N(11)-diethylnorspermine or with the polyamine biosynthesis inhibitor 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664). The comet assay showed that polyamine depletion resulted in DNA strand breaks. We also show that these DNA strand breaks occurred in cells where there was no expression of gamma-H2AX, which is a marker of DNA double-strand breaks. Thus, our conclusion is that polyamine depletion causes DNA single-strand breaks, which may be the cause for the observed delay in S phase progression.     

Molecular effects of lithium exposure during mouse and chick gastrulation and subsequent valve dysmorphogenesis

BACKGROUND: Lithium (Li) has been associated with cardiac teratogenicity in the developing fetus. We took advantage of the association of therapeutic administration of Li with an increase in heart defects to gain insight into both normal and pathological heart and valve development with GSK‐3 inhibition. The objective of this study was to define whether Li mimicry of canonical Wnt/β‐catenin signaling induces cardiac valve defects. METHODS: Li was administered by a single intraperitoneal injection to the pregnant mouse on embryonic day E6.75, much earlier than heretofore analyzed. On E15.5 developing heart defects were defined by Doppler ultrasound. The embryonic hearts were analyzed for changes in patterning of active canonical Wnt expression and nuclear factor of the activated T cells‐c1 (NFATc1), both key regulators of valve development. Li‐exposed chick embryos were used to define the early cell populations during gastrulation that are susceptible to GSK‐3 inhibition and may relate to valve formation. RESULTS: Li exposure during gastrulation decreased the number of prechordal plate (PP) cells that reached the anterior intestinal portal, a region associated with valve development. Li decreased expression of Hex, an endoderm cardiac inducing molecule, normally also expressed by the PP cells, and of Sox 4 at the anterior intestinal portal and NFAT, critical factors in valvulogenesis. CONCLUSIONS: Cells existing already during gastrulation are associated with valve formation days later. The Wnt/β‐catenin signaling in PP cells is normally repressed by Wnt antagonists and Hex is up‐regulated. The antagonism occurring at the receptor level is bypassed by Li exposure by its intracellular inactivation of GSK‐3 directly to augment Wnt signaling. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

A genetic variant of immunoglobulin γ2 is strongly associated with immunity to mucin 1 in patients with breast cancer

High levels of antibodies to mucin 1 (MUC1), a membrane-bound glycoprotein that is overexpressed in adenocarcinomas, are associated with good prognosis in patients with breast cancer. The aim of the present investigation was to determine whether GM and KM allotypes—genetic markers of IgG heavy chains and κ-type light chains, respectively—contribute to the magnitude of natural antibody responsiveness to MUC1 in patients with breast cancer. A total of 153 Caucasian subjects with breast cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. Anti-MUC1 IgG antibody levels in subjects who were carriers of the immunoglobulin γ2 allele GM 23 were significantly higher than in those who were noncarriers (P = 0.003). These results could potentially divide the population into high or low responders to MUC1, which has important implications for MUC1-based immunotherapeutic interventions in breast cancer.