Doxorubicin induces the persistent activation of intracellular transglutaminase 2 that protects from cell death

The activation of transglutaminase 2 (TG2), an enzyme that catalyzes post-translational modifications of proteins, has been implicated in apoptosis, cell adhesion and inflammatory responses. We previously reported that intracellular TG2 is activated under oxidative stress conditions, such as ultraviolet irradiation, ischemia-reperfusion, and hypoxia. In this study, we examined the effect of genotoxic stress on the intracellular activity of TG2 using doxorubicin which generates reactive oxygen species that lead to double-strand breakage of DNA. We demonstrated that doxorubicin elicits the persistent activation of TG2. Doxorubicin-induced TG2 activity was suppressed by treatment with caffeine at the early phase, N-acetylcysteine at the mid-phase, and EGTA at the late phase. However, treatment with a blocking antibody against TGFβ or toll-like receptor 2 showed no effect on TG2 activity, indicating that at least three different signaling pathways may be involved in the process of TG2 activation. In addition, using MEF cells defective for TG2 and cells overexpressing an activesite mutant of TG2, we revealed that doxorubicin-induced cell death is inversely correlated with TG2 activity. Our findings indicate that the persistent activation of TG2 by doxorubicin contributes to cell survival, suggesting that the mechanism-based inhibition of TG2 may be a novel strategy to prevent drug-resistance in doxorubicin treatment.     

Statistical method for prediction of gait kinematics with Gaussian process regression

We propose a novel methodology for predicting human gait pattern kinematics based on a statistical and stochastic approach using a method called Gaussian process regression (GPR). We selected 14 body parameters that significantly affect the gait pattern and 14 joint motions that represent gait kinematics. The body parameter and gait kinematics data were recorded from 113 subjects by anthropometric measurements and a motion capture system. We generated a regression model with GPR for gait pattern prediction and built a stochastic function mapping from body parameters to gait kinematics based on the database and GPR, and validated the model with a cross validation method. The function can not only produce trajectories for the joint motions associated with gait kinematics, but can also estimate the associated uncertainties. Our approach results in a novel, low-cost and subject-specific method for predicting gait kinematics with only the subject's body parameters as the necessary input, and also enables a comprehensive understanding of the correlation and uncertainty between body parameters and gait kinematics.     

Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

Electron-microscopic studies on the threshold value of calcium concentration for the release of storage granules and the acceleration of their degradation in the rat parathyroid gland

Summary To determine both a threshold value of calcium concentration (CC) for the release of storage granules and that for the acceleration of degradation of these granules, the rat parathyroid glands were perfused in situ with HEPES-Ringer solutions containing different concentration of Ca²⁺ for 10 min. With perfusates containing 0.83–1.21 mM Ca²⁺ (equivalent to 8–11 mg/dl serum calcium), the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged. With perfusates containing 0.83 mM Ca²⁺ (7.5 mg/dl) or less, however, both NSG-I and NSG-II decreased remarkably and the former was larger than the latter. On the contrary, with perfusates containing 1.27 mM Ca²⁺ (11.5 mg/dl) or more, NSG-II increased and the ratio of NSG-I to NSG-II was changed reversely. We concluded that a thereshold value of CC required for the release of storage granules may be present between 0.88 and 0.83 mM Ca²⁺ (8 and 7.5 mg/dl) and that a threshold value of CC for accelerating the transformation of type-I granules into type-II, the degradation of storage granules, may be situated at about 1.27 mM Ca²⁺ (11.5 mg/dl). Additionally, it was suggested that both prosecretory and storage granules are not only formed at the innermost Golgi cisterna but also at the trans-Golgi network.     

Mature ferredoxin-NADP reductase with a glutaminyl residue at N-terminus from spinach chloroplasts

When 35%-acetone extract of spinach chloroplasts was separated by SDS-PAGE, ferredoxin-NADP reductase (FNR) appeared as a single band at a molecular mass of 35 kDa. After the polypeptides on the SDS-PAGE plate were electroblotted onto PVDF membrane, the FNR band was cut out and analyzed for N-terminal structure in a gas-phase protein sequencer. Two different FNR peptides were identified: one with glutamine at its N-terminus (Gln-FNR) and the other with -pyroglutamic acid (tFNR) fraction was extracted from chloroplasts with their loosely bound FNR (lFNR) fraction removed in advance. The tFNR fraction contained Gln-FNR only. The Gln-FNR could be highly purified by affinity chromatography using a ferredoxin column. The purified Gln-FNR was digested with arginyl endopeptidase for peptide mapping and partial sequence analysis. Primary structure of Gln-FNR differed from that of lFNR loosely bound FNR - tFNR tightly bound FNR - -pyroglutamic acid at N-terminus     

Anuran metamorphosis: a model for gravitational study on motor development

Limbs and supporting structures of an organism experience a full weight of its own when it lands from water, because neutral buoyancy in the aquatic habitat will be no longer available in the terrestrial world. Metamorphosis of anuran amphibians presents a good research model to examine how this transition from non-loading to weight-loading affects development of motor capacity at the time of their first emergence on land. Our video analysis of the transitional anurans, Rana catesbeiana, at Gosner stage 46 (the stage of complete transformation) demonstrated that the take-off speed increased 1.23-fold after the first six hours of weight-loading on the wet ground. It did not increase further during the following three days of loading, and was close to the level of mature frogs with different body mass. During development of larvae in deep water with no chance of landing through metamorphosis, both tension and power of a hindlimb anti-gravity muscle increased 5-fold between stages 37 and 46. However, the muscle contractility increased more rapidly when the larvae could access the wet ground by their natural landing behavior after stages 41-42. Muscle power, one of major factors affecting locomotory speed, was 1.29-fold greater in the loaded than in the non-loaded larvae at the transitional stage. Thus, weight-loading had a potentially significant effect on the elevation of motor capacity, with a similar extent of increment in locomotory speed and muscle power during the last stages of metamorphosis. Such a motor adjustment of the froglets in a relatively short transitional period would be important for effective ecological interactions and survival in their inexperienced terrestrial life.     

Macrophagelike vacuolated renal tubular cells in the urine of a male with osmotic nephrosis associated with intravenous immunoglobulin therapy. A case report

BACKGROUND: Osmotic nephrosis is a form of renal tubular injury that has been found in patients treated with intravenous immunoglobulin (IVIG). CASE: A 46-year-old male who had two courses of chemotherapy for acute myelogenous leukemia was found to have refractory thrombocytopenia. After IVIG (Sandoglobulin 12%, Novartis) administration (1 g/kg) for five consecutive days, the patient became oliguric and eventually anuric on the fifth dose. Hemodialysis was initiated, and urine production was noted on day 2 of hospitalization. Routine cytologic examination of fresh, voided urine showed numerous macrophagelike, bland epithelial cells with abundant, multivacuolated cytoplasm. Cytokeratin immunostain revealed positivity, thus confirming the epithelial origin of these cells. CONCLUSION: To our knowledge, this is the first such case reported in the English-language cytology literature. Awareness of a patient's clinical history may be helpful in avoiding an incorrect diagnosis. Urine cytology may be useful in obtaining an early diagnosis of osmotic nephrosis in patients receiving high-dose IVIG therapy that may eliminate the need for a renal biopsy.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

Molecular modeling study for conformational changes of Sirtuin 2 due to substrate and inhibitor binding

Sirtuin is a member of NAD(+)-dependent deacetylase family. The structural details of Sirtuin 2 (SIRT2) complex will be very useful to discover the drug which might have beneficial effects on various diseases like cancer, diabetes, etc. Unfortunately, SIRT2 complex structure is not available yet, hence molecular docking was carried out to dock the substrate (NAD(+) and acetylated lysine) and inhibitor (sirtinol) in the NAD(+) binding site. The suitable binding orientation of substrate and inhibitor in the SIRT2 active site was selected and subjected to 5 ns molecular dynamics simulations to adjust the binding orientation of inhibitor and substrate as well as to identify the conformational changes in the active site. The result provides an insight about 3D SIRT2 structural details as well as the importance of F96 in deacetylation function. In addition, our simulations revealed the displacement of F96 upon substrate and inhibitor binding, inducing an extended conformation of loop3 and changing its interactions with the rest of SIRT2. We believe that our study could be helpful to gain a structural insight of SIRT2 and to design the receptor-based inhibitors.