Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma

Summary Epigenetic models for tumor formation assume that oncogenic transformation results from changes in the activity of otherwise normal genes. Since gene activity can be inhibited by DNA methylation, and inactivation of tumor suppressor genes is a fundamental process in oncogenesis, we investigated the methylation status of the retinoblastoma suppressor gene (RB gene) on chromosome 13, in blood and tumor cells from 21 retinoblastoma patients. Using methylation-sensitive restriction enzymes and a cloned DNA probe for the unmethylated CpG island at the 5 end of RB gene, we obtained evidence of hypermethylation of this gene in a sporadic unilateral retinoblastoma tumor. The closely linked esterase D gene and a CpG-rich island on chromosome 15 were not affected. We suggest that changes in the methylation pattern of the RB gene play a role in the development and spontaneous regression of some retinoblastoma tumors.     

A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding.

To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.     

Activated platelets contribute importantly to myocardial reperfusion injury

Platelets become activated during myocardial infarction (MI), but the direct contribution of activated platelets to myocardial reperfusion injury in vivo has yet to be reported. We tested the hypothesis that activated platelets contribute importantly to reperfusion injury during MI in mice. After 30 min of ischemia and 60 min of reperfusion, P-selectin knockout mice had a significantly smaller infarct size than that of wild-type mice (P < 0.05). Platelets were detected by P-selectin antibody in the previously ischemic region of wild-type mice as early as 2 min postreperfusion after 45 min, but not 20 min, of ischemia. The appearance of neutrophils in the heart was delayed when compared with platelets. Flow cytometry showed that the number of activated platelets more than doubled after 45 min of ischemia when compared with 20 min of ischemia or sham treatment (P < 0.05). Platelet-rich or platelet-poor plasma was then transfused from either sham-operated or infarcted mice after 45 and 10 min of ischemia-reperfusion to mice undergoing 20 and 60 min of ischemia-reperfusion. Infarct size was increased by threefold and platelet accumulation was remarkably enhanced in mice treated with wild-type, MI-activated platelet-rich plasma but not in mice receiving either platelet-poor plasma from wild types or MI-activated platelet-rich plasma from P-selectin knockout mice. In conclusion, circulating platelets become activated early during reperfusion and their activation depends on the duration of the preceding coronary occlusion and is proportional to the extent of myocardial injury. Activated platelets play an important role in the process of myocardial ischemia-reperfusion injury, and platelet-derived P-selectin is a critical mediator.     

Oxidative stress-induced calcium signaling in Arabidopsis

Many environmental stresses result in increased generation of active oxygen species in plant cells. This leads to the induction of protective mechanisms, including changes in gene expression, which lead to antioxidant activity, the recovery of redox balance, and recovery from damage/toxicity. Relatively little is known about the signaling events that link perception of increased active oxygen species levels to gene expression in plants. We have investigated the role of calcium signaling in H2O2-induced expression of the GLUTATHIONE-S-TRANSFERASE1 (GST1) gene. Challenge with H2O2 triggered a biphasic Ca2+ elevation in Arabidopsis seedlings. The early Ca2+ peak localized to the cotyledons, whereas the late Ca2+ rise was restricted to the root. The two phases of the Ca2+ response were independent of each other, as shown by severing shoot from root tissues before H2O2 challenge. Modulation of the height of Ca2+ rises had a corresponding effect upon H2O2-induced GST1 expression. Application of the calcium channel blocker lanthanum reduced the height of the first Ca2+ peak and concomitantly inhibited GST1 expression. Conversely, enhancing the height of the H2O2-triggered Ca2+ signature by treatment with L-buthionine-[S,R]-sulfoximine (an inhibitor of glutathione synthesis) lead to enhancement of GST1 induction. This finding also indicates that changes in the cellular redox balance constitute an early event in H2O2 signal transduction as reduction of the cellular redox buffer and thus the cell's ability to maintain a high GSH/GSSG ratio potentiated the plant's antioxidant response.     

Shear stress-induced binding of von willebrand factor to platelets

Shear stress-induced platelet aggregation requires von Willebrand factor (vWF), platelet glycoprotein (GP) Ib, GPIIb-IIIa, Ca²⁺, and adenosine diphosphate (ADP). Recent reports using vWF labeled with either ¹²⁵I or fluorescein isothiocyanate (FITC) have demonstrated that in shear-fields, vWF binds to both GPIb and GPHb-IIIa. The sequence of the vWF binding to the two platelet receptors has not been precisely determined in these reports. In this study, a flow cytometry technique using a primary anti-vWF antibody and a secondary FITC IgG antibody was used to measure shear stress-induced vWF binding to platelets. Washed normal platelets suspended at 50,000/μl with purified large VWF multimers were exposed to laminar shear stresses of 15 to 120 dynes/cm² for 30 sec. At this low platelet count, little or no aggregation occurred in the shear fields. A significant increase in post-shear vWF-positive platelets was consistently observed. Experiments with platelets from normal and severe von Willebrand's disease (vWD) (which lack plasma and platelet α-granule vWF) demonstrated that exogenous vWF predominately contributed to the platelet-vWF binding. Blockade of platelet GPIb with the monoclonal anti-GPIb antibody, 6D1, completely inhibited shear stress-induced platelet-vWF attachment. In contrast, blockade of GPIIb-IIIa with monoclonal anti-GPIIb-IIIa antibodies, 10E5 or c7E3, or with the GPIIb-IIIa-blocking tetrapeptide, RGDS, had little or no inhibitory effect on platelet-vWF binding. These data demonstrate that the binding of vWF to GPIb is likely to be the initial shear-induced platelet-ligand binding event. © 1997 Elsevier Science Ltd     

Stress-induced acidification may contribute to formation of unusual structures in C9orf72-repeats

Background

Expansion of the C9orf72 hexanucleotide repeat (GGGGCC)_n·(GGCCCC)_n is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both strands of the C9orf72 repeat have been shown to form unusual DNA and RNA structures that are thought to be involved in mutagenesis and/or pathogenesis. We previously showed that the C-rich DNA strands from the C9orf72 repeat can form four-stranded quadruplexes at neutral pH. The cytosine residues become protonated under slightly acidic pH (pH?4.5–6.2), facilitating the formation of intercalated i-motif structures.

Recycling endosomes contribute to autophagosome formation

Autophagosome formation is a complex cellular process, which requires major membrane rearrangements leading to the creation of a relatively large double-membrane vesicle that directs its contents to the lysosome for degradation. Although various membrane compartments have been identified as sources for autophagosomal membranes, the molecular mechanism underlying these membrane trafficking steps remains elusive. To address this question we performed a systematic analysis testing all known Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins for their ability to inhibit autophagosome formation by disrupting a specific membrane trafficking step. TBC proteins are thought to act as inhibitors of Rab GTPases, which regulate membrane trafficking events. Up to 11 TBC proteins inhibit autophagy when overexpressed and one of these, TBC1D14, acts at an early stage during autophagosome formation and is involved in regulating recycling endosomal traffic. We found that the early acting autophagy proteins ATG9 and ULK1 localize to transferrin receptor (TFR)-positive recycling endosomes (RE), which are tubulated by excess TBC1D14 leading to an inhibition of autophagosome formation. Finally, transferrin (TF)-containing recycling endosomal membranes can be incorporated into newly forming autophagosomes, although it is likely that most of the autophagosome membrane is subsequently acquired from other sources.     

Recycling endosomes contribute to autophagosome formation

Autophagosome formation is a complex cellular process, which requires major membrane rearrangements leading to the creation of a relatively large double-membrane vesicle that directs its contents to the lysosome for degradation. Although various membrane compartments have been identified as sources for autophagosomal membranes, the molecular mechanism underlying these membrane trafficking steps remains elusive. To address this question we performed a systematic analysis testing all known Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins for their ability to inhibit autophagosome formation by disrupting a specific membrane trafficking step. TBC proteins are thought to act as inhibitors of Rab GTPases, which regulate membrane trafficking events. Up to 11 TBC proteins inhibit autophagy when overexpressed and one of these, TBC1D14, acts at an early stage during autophagosome formation and is involved in regulating recycling endosomal traffic. We found that the early acting autophagy proteins ATG9 and ULK1 localize to transferrin receptor (TFR)-positive recycling endosomes (RE), which are tubulated by excess TBC1D14 leading to an inhibition of autophagosome formation. Finally, transferrin (TF)-containing recycling endosomal membranes can be incorporated into newly forming autophagosomes, although it is likely that most of the autophagosome membrane is subsequently acquired from other sources.     

Oxidative stress-related mechanisms affecting response to aspirin in diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a major cardiovascular risk factor. Persistent platelet activation plays a key role in atherothrombosis in T2DM. However, current antiplatelet treatments appear less effective in T2DM patients vs nondiabetics at similar risk. A large body of evidence supports the contention that oxidative stress, which characterizes DM, may be responsible, at least in part, for less-than-expected response to aspirin, with multiple mechanisms acting at several levels. This review discusses the pathophysiological mechanisms related to oxidative stress and contributing to suboptimal aspirin action or responsiveness. These include: (1) mechanisms counteracting the antiplatelet effect of aspirin, such as reduced platelet sensitivity to the antiaggregating effects of NO, due to high-glucose-mediated oxidative stress; (2) mechanisms interfering with COX acetylation especially at the platelet level, e.g., lipid hydroperoxide-dependent impaired acetylating effects of aspirin; (3) mechanisms favoring platelet priming (lipid hydroperoxides) or activation (F₂-isoprostanes, acting as partial agonists of thromboxane receptor), or aldose-reductase pathway-mediated oxidative stress, leading to enhanced platelet thromboxane A₂ generation or thromboxane receptor activation; (4) mechanisms favoring platelet recruitment, such as aspirin-induced platelet isoprostane formation; (5) modulation of megakaryocyte generation and thrombopoiesis by oxidative HO-1 inhibition; and (6) aspirin–iron interactions, eventually resulting in impaired pharmacological activity of aspirin, lipoperoxide burden, and enhanced generation of hydroxyl radicals capable of promoting protein kinase C activation and platelet aggregation. Acknowledgment of oxidative stress as a major contributor, not only of vascular complications, but also of suboptimal response to antiplatelet agents in T2DM, may open the way to designing and testing novel antithrombotic strategies, specifically targeting oxidative stress-mediated mechanisms of less-than-expected response to aspirin.     

Oxidative stress-induced calcium signalling in Aspergillus nidulans

The effects of oxidative stress on levels of calcium ion (Ca(2+)) in Aspergillus nidulans were measured using strains expressing aequorin in the cytoplasm (Aeq(cyt)) and mitochondria (Aeq(mt)). When oxidative stress was induced by exposure to 10-mM H(2)O(2), the mitochondrial calcium response (Ca(mt)(2+)) was greater than the change in cytoplasmic calcium (Ca(c)(2+)). The Ca(mt)(2+) response to H(2)O(2) was dose dependent, while the increase in [Ca(c)(2+)] did not change with increasing H(2)O(2). The increase in both [Ca(c)(2+)] and [Ca(mt)(2+)] in response to oxidative stress was enhanced by exposure of cells to Ca(2+). The presence of chelator in the external medium only partially inhibited the Ca(mt)(2+) and Ca(c)(2+) responses to oxidative stress. Reagents that alter calcium fluxes had varied effects on the Ca(mt)(2+) response to peroxide. Ruthenium red blocked the increase in [Ca(mt)(2+)], while neomycin caused an even greater increase in [Ca(mt)(2+)]. Treatment with ruthenium red and neomycin had no effect on the Ca(c)(2+) response. Bafilomycin A and oligomycin had no effect on either the mitochondrial or cytoplasmic response. Inhibitors of both voltage-regulated calcium channels and intracellular calcium release channels inhibited the Ca(2+)-dependent component of the Ca(mt)(2+) response to oxidative stress. We conclude that the more significant Ca(2+) response to oxidative stress occurs in the mitochondria and that both intracellular and extracellular calcium pools can contribute to the increases in [Ca(c)(2+)] and [Ca(mt)(2+)] induced by oxidative stress.     

Oxidative stress-induced insulin resistance in rat skeletal muscle: role of glycogen synthase kinase-3

Oxidative stress can contribute to the multifactorial etiology of whole body and skeletal muscle insulin resistance. No investigation has directly assessed the effect of an in vitro oxidant stress on insulin action in intact mammalian skeletal muscle. Therefore, the purpose of the present study was to characterize the molecular actions of a low-grade oxidant stress (H(2)O(2)) on insulin signaling and glucose transport in isolated skeletal muscle of lean Zucker rats. Soleus strips were incubated in 8 mM glucose for 2 h in the absence or presence of 100 mU/ml glucose oxidase, which produces H(2)O(2) at approximately 90 microM. By itself, H(2)O(2) significantly (P < 0.05) activated basal glucose transport activity, net glycogen synthesis, and glycogen synthase activity and increased phosphorylation of insulin receptor (Tyr), Akt (Ser(473)), and GSK-3beta (Ser(9)). In contrast, this oxidant stress significantly inhibited the expected insulin-mediated enhancements in glucose transport, glycogen synthesis, and these signaling factors and allowed GSK-3beta to retain a more active form. In the presence of CT-98014, a selective GSK-3 inhibitor, the ability of insulin to stimulate glucose transport and glycogen synthesis during exposure to this oxidant stress was enhanced by 20% and 39% (P < 0.05), respectively, and insulin stimulation of the phosphorylation of insulin receptor, Akt, and GSK-3 was significantly increased by 36-58% (P < 0.05). These results indicate that an oxidant stress can directly and rapidly induce substantial insulin resistance of skeletal muscle insulin signaling, glucose transport, and glycogen synthesis. Moreover, a small, but significant, portion of this oxidative stress-induced insulin resistance is associated with a reduced insulin-mediated suppression of the active form of GSK-3beta.     

Multiple avirulence paralogues in cereal powdery mildew fungi may contribute to parasite fitness and defeat of plant resistance

Powdery mildews, obligate biotrophic fungal parasites on a wide range of important crops, can be controlled by plant resistance (R) genes, but these are rapidly overcome by parasite mutants evading recognition. It is unknown how this rapid evolution occurs without apparent loss of parasite fitness. R proteins recognize avirulence (AVR) molecules from parasites in a gene-for-gene manner and trigger defense responses. We identify AVR(a10) and AVR(k1) of barley powdery mildew fungus, Blumeria graminis f sp hordei (Bgh), and show that they induce both cell death and inaccessibility when transiently expressed in Mla10 and Mlk1 barley (Hordeum vulgare) varieties, respectively. In contrast with other reported fungal AVR genes, AVR(a10) and AVR(k1) encode proteins that lack secretion signal peptides and enhance infection success on susceptible host plant cells. AVR(a10) and AVR(k1) belong to a large family with >30 paralogues in the genome of Bgh, and homologous sequences are present in other formae speciales of the fungus infecting other grasses. Our findings imply that the mildew fungus has a repertoire of AVR genes, which may function as effectors and contribute to parasite virulence. Multiple copies of related but distinct AVR effector paralogues might enable populations of Bgh to rapidly overcome host R genes while maintaining virulence.     

Sequence variability of Borna disease virus: resistance to superinfection may contribute to high genome stability in persistently infected cells

The RNA genome of Borna disease virus (BDV) shows extraordinary stability in persistently infected cell cultures. We performed bottleneck experiments in which virus populations from single infected cells were allowed to spread through cultures of uninfected cells and in which RNase protection assays were used to identify virus variants with mutations in a 535-nucleotide fragment of the M-G open reading frames. In one of the cell cultures, the major virus species (designated 2/1) was a variant with two point mutations in the G open reading frame. When fresh cells were infected with a low dose of a virus stock prepared from 2/1-containing cells, only a minority of the resulting persistently infected cultures contained detectable levels of the variant, whereas the others all seemed to contain wild-type virus. The BDV variant 2/1 remained stable in the various persistently infected cell cultures, indicating that the cells were resistant to superinfection by wild-type virus. Indeed, cells persistently infected with prototype BDV He/80 were also found to resist superinfection with strain V and vice versa. Our screen for mutations in the viral M and G genes of different rat-derived BDV virus stocks revealed that only one of four stocks believed to contain He/80 harbored virus with the original sequence. Two stocks mainly contained a novel virus variant with about 3% sequence divergence, whereas the fourth stock contained a mixture of both viruses. When the mixture was inoculated into the brains of newborn mice, the novel variant was preferentially amplified. These results provide evidence that the BDV genome is mutating more frequently than estimated from its invariant appearance in persistently infected cell cultures and that resistance to superinfection might strongly select against novel variants.     

Postprandial recruitment of neutrophils may contribute to endothelial dysfunction

Atherosclerosis is a low-grade inflammatory disease involving leukocytes, lipids, and glucose leading to endothelial dysfunction. Since activation of neutrophils by triglycerides and glucose has been described in vitro, we hypothesized that the postprandial phase is an inflammatory state affecting leukocytes, possibly contributing to endothelial dysfunction. We measured postprandial blood leukocyte counts, cytokines, hydroperoxides (HPOs), and flow-mediated vasodilation (FMD) in eight healthy males (age 23 +/- 2 years) after a FAT (50 g/m2) and GLUCOSE challenge (37.5 g/m2), a combination of both (MIXED test), and after WATER. All tests, except WATER, resulted in significantly impaired FMD (10% reduction) between t = 1 h and t = 3 h, accompanied by a significant increase of neutrophils (59% after FAT and 28% after GLUCOSE and MIXED), total plasma HPOs (15 to 31% increase), and plasma interleukin-8 (IL-8) (50-130% increase). WATER did not affect FMD, neutrophils, HPOs, or IL-8. Lymphocytes increased gradually in all tests (40-70% increase at t = 10 h compared with t = 0; P < 0.005), paralleling a gradual 3- to 5-fold interleukin-6 increase. Monocyte and erythrocyte counts did not change in any test. In conclusion, the neutrophil increment during postprandial lipemia and glycemia with concomitant IL-8 and HPO increases may contribute to endothelial dysfunction. Lymphocyte increment is a nonspecific diurnal process. Postprandial intravascular inflammatory changes may be relevant for the pathogenesis of atherosclerosis.     

Molecular mimicry may contribute to pathogenesis of ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with mucosal inflammation and ulceration of the colon. There seems to be no single etiological factor responsible for the onset of the disease. Autoimmunity has been emphasized in the pathogenesis of UC. Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) are common in UC, and recently two major species of proteins immunoreactive to pANCA were detected in bacteria from the anaerobic libraries. This implicates colonic bacterial protein as a possible trigger for the disease-associated immune response. Autoantibodies and T-cell response against human tropomyosin isoform 5 (hTM5), an isoform predominantly expressed in colon epithelial cells, were demonstrated in patients with UC but not in Crohn's colitis. We identified two bacterial protein sequences in NCBI database that have regions of significant sequence homology with hTM5. Our hypothesis is that molecular mimicry may be responsible for the pathogenesis of UC.     

Free radicals may contribute to oxidative skeletal muscle fatigue

We used mouse soleus in vitro (n = 30) and canine gastrocnemius-plantaris preparations (n = 20) pump-perfused at the animal's blood pressure to establish if free radicals contribute to fatigue in oxidative skeletal muscle. The soleus from each leg contracted for 200 ms (70 Hz) once every minute for 60 min in Hepes buffer gassed with 100% oxygen at 27 degrees C. When contracting in Hepes alone, both muscles fatigued at 0.9 mN/mm2.min over the 60 min. The addition of purines to the bath increased the rate to 1.4 mN/mm2.min and the addition of xanthine oxidase to generate free radicals increased the rate again to 1.9 mN/mm2.min. Thus free radicals appeared to attenuate oxidative skeletal muscle function. Each canine muscle contracted isometrically at 4 Hz for 30 min and then rested for 45 min before contracting for a second 30 min at 4 Hz. In each experiment, we infused saline at 0.76 mL/min into resting muscle and at 1.91 mL/min during the first contraction period. During the remainder of the experiment, we infused, at the same rates, saline (n = 4), 10 microM dimethyl sulfoxide (DMSO) (n = 4) to identify the effect of scavenging hydroxyl radicals, 1 mM allopurinol to establish the effect of blocking xanthine oxidase (n = 4), or 200 microM desferoxamine to determine the effect of chelating iron (n = 4). With saline, the fatigue rate over the 30 min of contractions increased from 5.0 +/- 0.2 to 6.3 +/- 0.5 N/kg.min from the first to the second stimulation period. The fatigue rate was slower in the second period with each of the three experimental substances (DMSO, 5.9 +/- 0.8 to 3.2 +/- 0.3; allopurinol, 7.3 +/- 1.1 to 4.6 +/- 0.6; desferoxamine, 6.8 +/- 0.8 to 4.4 +/- 0.8 N/kg.min). The fatigue rate was the same as control when DMSO was infused only during the second contraction period. Therefore, free radicals appeared to contribute to fatigue in oxidative skeletal muscle.     

Replication-associated purine asymmetry may contribute to strand-biased gene distribution

Among prokaryotic genomes, the distribution of genes on the leading and lagging strands of the replication fork is known to be biased. Several hypotheses explaining this strand-biased gene distribution (SGD) have been proposed, but none have been tested or supported by sufficient data analyses. In this work we have analyzed 211 prokaryotic genomes in terms of compositional strand asymmetries and the presence or absence of polC and have found that SGD correlates not only with polC, but also with purine asymmetry (PAS). Furthermore, SGD, PAS, and polC are all features associated with a group of low-GC, gram-positive bacteria (Firmicutes). We conclude that PAS is a characteristic of organisms with a heterodimeric DNA polymerase III alpha-subunit constituted by polC and dnaE, which may play a direct role in the maintenance of SGD.     

Maturational differences in chlorpyrifos-oxonase activity may contribute to age-related sensitivity to chlorpyrifos

Chlorpyrifos (CPF), a commonly used cholinesterase-inhibiting insecticide, is lethal at much lower doses to young animals than adults. To explain this higher sensitivity in younger animals, we hypothesized that young rats have less chlorpyrifos-oxonase (CPFOase) activity than adults. To test this hypothesis, CPFOase activity was measured in the brain, plasma, and liver of male, postnatal day 4 (PND4) and adult (PND90) Long-Evans rats. CPFOase is biochemically defined as a Ca²⁺-dependent A-esterase that hydrolyzes chlorpyrifos-oxon (CPFO), the active metabolite of CPF. No brain CPFOase activity was detected at either age. Plasma and liver CPFOase activities were markedly lower at PND4 compared to adult: PND4 plasma and liver CPFOase activities were 1/11 and 1/2 the adult plasma and liver activities, respectively. Because the K_m of CPFOase activity was high (i.e., 210–380 μM), it was important to determine if this CPFOase activity could hydrolyze physiologically relevant concentrations (i.e., nM to low μM) of CPFO. This was accomplished by comparing the shifts in the tissue acetylcholinesterase (AChE) IC₅₀ for CPFO in the presence or absence of CPFOase activity. One would expect an increase in the “apparent” IC₅₀ if CPFOase hydrolyzes substantial amounts of CPFO during the 30 minutes the tissue is preincubated with the CPFO. In the adult, both plasma and liver AChE apparent IC₅₀ values were higher in the presence of CPFOase activity, suggesting that the CPFOase in those tissues was capable of hydrolyzing physiologically relevant concentrations of CPFO within 30 minutes. In young animals, however, there was less of a shift in the IC₅₀ curves compared to the adult, confirming that the young animal has less capacity than the adult to detoxify physiologically relevant concentrations of CPFO via CPFOase. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 279–287, 1997.