A Three Dimensional Model of Human Thiopurine Methyltransferase; Ligand Interactions and Structural Consequences of Naturally Occurring Mutations

The three-dimensional model of human thiopurine methyltransferase (hTPMT) was constructed by molecular modeling. A multiple alignment of AdoMet dependent methyltransferases based on a structural superposition of the AdoMet binding domain of Hhai, TaqI and rCOMT was used in the modeling procedure. The reliability of the model was examined by comparing its conformation and packing properties with those of Hhai, TaqI and rCOMT and structures in the PDB-database. The examined criteria indicated a reliable model structure. The model gave insight into the structural effects of naturally occurring mutations of the hTPMT allele, and was used to characterize the ligand interactions of the protein. The residues Gln42 and Glu91 were predicted to participate in AdoMet binding through H-bond interactions whereas Phe146 participates through Van der Waal interaction. The cationic methyl-sulphonium group of AdoMet was located close to the aromatic residue Phe40. The model also indicated that substrates interact with hTPMT situated in a pocket consisting of the hydrophobic residues Phe40, Met148, Val184, Val220 and the charged residues Lys145, Glu218, Lys219. These residues were also included in a predictive explanation for the inhibitor/substrate preference of the enzyme. The most frequent of naturally occurring mutations was predicted to cause alterations on the surface of the protein with minor/none structural consequences. The mutation Ala80-Pro seemed directly to cause an inactive enzyme by disrupting the structure of the binding site of AdoMet.Electronic Supplementary Material available.     

uPAR Expression Pattern in Patients with Urothelial Carcinoma of the Bladder – Possible Clinical Implications

The objective of the present study was to confirm the expression and localisation pattern of the urokinase-type plasminogen activator receptor (uPAR) focusing on its possible clinical relevance in patients with urothelial neoplasia of the bladder. uPAR is a central molecule in tissue remodelling during cancer invasion and metastasis and is an established prognostic marker in various cancer diseases other than bladder cancer. Formalin-fixed and paraffin-embedded tumour-tissue blocks from 186 patients treated with radical cystectomy were analysed. uPAR expression was scored as either negative or positive as well as by the actual score. Separate scores were obtained for cancer cells, macrophages and myofibroblasts at the invasive front and in tumour core. We were able to confirm, in an independent patient cohort, the tissue expression and localisation pattern of uPAR as investigated by Immunohistochemistry as well as a significant association between uPAR positivity and increasing tumour stage and tumour grade. This demonstrates the robustness of our previous and current findings. In addition the association between uPAR positive myofibroblasts and poor survival was reproduced. The highest hazard ratios for survival were seen for uPAR positive myofibroblasts both at the invasive front and in tumour core. Evaluating uPAR expression by the actual score showed a significant association between uPAR positive myofibroblasts in tumour core and an increased risk of cancer specific mortality. Our investigations have generated new and valuable biological information about the cell types being involved in tumour invasion and progression through the plasminogen activation system.     

Annual variation in lymphocystis infection frequency in flounder, Platichthys flesus (L.)

The infection frequency of the virus induced lymphocystis disease has been registered in flounder from the Oslofjord, Norway, during a 4 year period. The disease has a clear regular seasonal variation, and also an annual variation. The percentage infection in the summer was 1–10%, and in the winter up to 57%. The possibility that the high pollution level of the inner Oslofjord could be the reason for the high frequency of lymphocystis disease, is discussed.     

Binding of S-adenosylhomocysteine to various domains of the plasma membrane and to the endoplasmic reticulum from rat liver: Relation between binding and phospholipid methyltransferase activity

S-Adenosylhomocysteine (AdoHcy) binding to various membrane fractions of rat liver was determined at pH 7.4, using an oil centrifugation technique. The highest binding activity was found in the heavy microsomal (M-H) fraction enriched in endoplasmic reticulum, but high binding activity was also observed in the light microsomal fractions enriched in blood sinusoidal membranes (M-L fraction), and the heavy nuclear fraction (N-H fraction) containing the contiguous area. A substantial portion of AdoHcy binding activity in the M-L fraction may be ascribed to contamination of this fraction with endoplasmic reticulum, as indicated by the distribution of NADPH cytochrome c reductase activity. Binding activity was low in the light nuclear (N-L) fraction corresponding to the bile canaliculi. Phospholipid methyltransferase activity was determined in the same membrane fractions under similar conditions (pH 7.4), and in the absence and presence of added phospholipids. The distribution of the enzyme activity was dependent on the presence of exogenous phospholipids, and grossly similar to AdoHcy binding, the highest activities being observed in the M-H and the M-L fractions. The N-H fraction, rich in AdoHcy-binding activity, demonstrated, however, a very low phospholipid methyltransferase activity. It is concluded that AdoHcy-binding activity is not confined to the plasma membranes, and a major fraction of the binding activity resides on membranes derived from the endoplasmic reticulum. Also, the present results add to previous data suggesting that phospholipid methyltransferase does not totally account for the AdoHcy-binding sites on rat liver membranes.     

Short‐term insurance versus long‐term bet‐hedging strategies as adaptations to variable environments

Understanding how organisms adapt to environmental variation is a key challenge of biology. Central to this are bet‐hedging strategies that maximize geometric mean fitness across generations, either by being conservative or diversifying phenotypes. Theoretical models have identified environmental variation across generations with multiplicative fitness effects as driving the evolution of bet‐hedging. However, behavioral ecology has revealed adaptive responses to additive fitness effects of environmental variation within lifetimes, either through insurance or risk‐sensitive strategies. Here, we explore whether the effects of adaptive insurance interact with the evolution of bet‐hedging by varying the position and skew of both arithmetic and geometric mean fitness functions. We find that insurance causes the optimal phenotype to shift from the peak to down the less steeply decreasing side of the fitness function, and that conservative bet‐hedging produces an additional shift on top of this, which decreases as adaptive phenotypic variation from diversifying bet‐hedging increases. When diversifying bet‐hedging is not an option, environmental canalization to reduce phenotypic variation is almost always favored, except where the tails of the fitness function are steeply convex and produce a novel risk‐sensitive increase in phenotypic variance akin to diversifying bet‐hedging. Importantly, using skewed fitness functions, we provide the first model that explicitly addresses how conservative and diversifying bet‐hedging strategies might coexist.     

Expression of bone morphogenetic protein 4 and its receptors in the remodeling heart

Aims

Heart failure is associated with activation of fetal gene programs. Bone morphogenetic proteins (BMPs) regulate embryonic development through interaction with BMP receptors (BMPRs) on the cell surface. We investigated if the expression of BMP4 and its receptors BMPR1a and BMPR2 were activated in post-infarction remodeling and heart failure.

Main methods

Left ventricular biopsies were taken from explanted hearts of patients with end-stage heart failure due to dilated cardiomyopathy (CMP; n = 15) or ischemic heart disease (CAD; n = 9), and compared with homograft control preparations from organ donors deceased due to non-cardiac causes (n = 7). Other samples were taken from patients undergoing coronary artery bypass grafting (CABG; n = 11). Mice were subjected to induced infarction by permanent coronary artery ligation or sham operation, and hearts were sampled serially thereafter (n = 7 at each time point).

Key findings

Human and mouse hearts expressed BMP4 and both receptor subtypes. CABG and CMP patients had increased expression of mRNA encoding for BMP4, but unchanged protein. Mouse hearts had increased BMP4 precursor protein 24 h after infarction. BMPR1a protein decreased in CAD patients and initially in postinfarcted mouse hearts, but increased again in the latter after two weeks. Human recombinant BMP4 promoted survival after H₂O₂ injury in HL-1 cells, and also protected adult mouse cardiomyocytes against hypoxia–reoxygenation injury.

Significance

Adult hearts express BMP4, the mRNA increasingly so in patients with coronary artery disease with good cardiac function. BMPRs are downregulated in cardiac remodeling and failure. Recombinant BMP4 has protective effects on cultured cardiomyocytes.     

The hunchback and its neighbours: proline as an environmental modulator.

The unique ability of Pro or Pro-rich repeats to affect the stability and function of proteins has recently been highlighted by biophysical studies on fragments from prions, signalling domains and muscle proteins. Pro-rich regions have been observed to either occupy disordered states or adopt various helical structures; some are also able to undergo an environmental-dependent transformation between these states. Such a transformation could explain some of the inherent functional properties of the parent proteins and, additionally, can be efficiently exploited to generate novel temperature- and pH-switches in more conventional globular proteins.     

Impacts of extreme winter warming events on plant physiology in a sub‐Arctic heath community

Insulation provided by snow cover and tolerance of freezing by physiological acclimation allows Arctic plants to survive cold winter temperatures. However, both the protection mechanisms may be lost with winter climate change, especially during extreme winter warming events where loss of snow cover from snow melt results in exposure of plants to warm temperatures and then returning extreme cold in the absence of insulating snow. These events cause considerable damage to Arctic plants, but physiological responses behind such damage remain unknown. Here, we report simulations of extreme winter warming events using infrared heating lamps and soil warming cables in a sub‐Arctic heathland. During these events, we measured maximum quantum yield of photosystem II (PSII), photosynthesis, respiration, bud swelling and associated bud carbohydrate changes and lipid peroxidation to identify physiological responses during and after the winter warming events in three dwarf shrub species: Empetrum hermaphroditum, Vaccinium vitis‐idaea and Vaccinium myrtillus. Winter warming increased maximum quantum yield of PSII, and photosynthesis was initiated for E. hermaphroditum and V. vitis‐idaea. Bud swelling, bud carbohydrate decreases and lipid peroxidation were largest for E. hermaphroditum, whereas V. myrtillus and V. vitis‐idaea showed no or less strong responses. Increased physiological activity and bud swelling suggest that sub‐Arctic plants can initiate spring‐like development in response to a short winter warming event. Lipid peroxidation suggests that plants experience increased winter stress. The observed differences between species in physiological responses are broadly consistent with interspecific differences in damage seen in previous studies, with E. hermaphroditum and V. myrtillus tending to be most sensitive. This suggests that initiation of spring‐like development may be a major driver in the damage caused by winter warming events that are predicted to become more frequent in some regions of the Arctic and that may ultimately drive plant community shifts.