Prospective monitoring of the Epstein–Barr virus DNA by a real-time quantitative polymerase chain reaction after allogenic stem cell transplantation

Epstein-Barr virus (EBV)-related lymphoproliferative disorder (LPD) is a serious complication of haematopoietic stem cell transplantation (HSCT). To clarify the frequency, natural course and risk factors for LPD, we prospectively monitored 38 allogeneic (allo)-HSCT patients, focusing on the use of anti-thymocyte globulin (ATG). We used a recently developed real-time polymerase chain reaction assay to monitor EBV genome load. The subjects consisted of 19 patients given ATG for conditioning and 19 patients not given ATG. Of the 19 patients given ATG, 47.4% (nine patients) had a significant increase in EBV genome load (10(2.5) copies/microg DNA). Of these nine patients, two developed LPD. Therefore, 10.5% of the patients receiving allo-HSCT with ATG developed LPD. In contrast, none of the 19 patients without ATG had a significantly increased EBV load. The increases in viral load were observed in the second or third month after HSCT. We found that the peak viral loads of LPD patients were > 10(4.0 ) copies/microg DNA. On the other hand, the viral loads of most patients with no symptoms were < 10(2.5) copies/microg DNA. In conclusion, routine monitoring of EBV load during the second and third months after transplantation may benefit patients undergoing HSCT with ATG. We propose that an EBV load > 10(2.5) copies/microg DNA is the reactivation of EBV, and that an EBV load > 10(4.0) copies/microg DNA is indicative of developing LPD.     

Antiepileptic drug therapy for adult patients with intractable seizures]

Epileptic syndrome, antiepileptic drug (AED) therapy and mental disorder were studied in 223 patients with intractable epilepsy, who were admitted to our epilepsy center between 1992 and 2000. Symptomatic localization-related epilepsy was diagnosed in 86.1% of patients, symptomatic or cryptogenic generalized epilepsy in 7.6%, idiopathic generalized epilepsy in 1.8%, unclassifiable epilepsy in 3.1% and non-epilepsy in 1.3% on discharge. Only 6.3% had diagnoses on discharge that were incongruent with their diagnoses on admission. AED therapy during admission improved markedly in 50% of patients and moderately in 20%, however, 60% had seizures more frequently than 4 a month on discharge. Generalized tonic and clonic seizures were suppressed completely in 82.5% of patients. The number of AEDs used were 2 AEDs in 28.6%, 3 in 39.1% and 4 in 22.3%. Only 6.4% of patients were on monotherapy on discharge. Mental retardation was in 58.7%, schizophrenia-like psychosis in 8.5%, delusional disorder in 1.8%, mood disorder in 3.6%, AED-related disorder in 14.3% and psychogenic disorders in 21.5%. AED therapy is effective for intractable seizures, but it is limited in its effect. Mental disorders also coexisted in most of patients. Therefore comprehensive therapy of epilepsy is necessary for patients with intractable seizures.     

Effects of Volatile Anesthetics on N-methyl-d-aspartate Excitotoxicity in Primary Rat Neuronal-Glial Cultures

Background: Volatile anesthetics are known to ameliorate experimental ischemic brain injury. A possible mechanism is inhibition of excitotoxic cascades induced by excessive glutamatergic stimulation. This study examined interactions between volatile anesthetics and excitotoxic stress.

Methods: Primary cortical neuronal-glial cultures were exposed to N-methyl-d-aspartate (NMDA) or glutamate and isoflurane (0.1-3.3 mm), sevoflurane (0.1-2.9 mm), halothane (0.1-2.9 mm), or 10 [mu]m (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801). Lactate dehydrogenase release was measured 24 h later. In other cultures, effects of volatile anesthetics on Ca++ uptake and mitochondrial membrane potential were determined in the presence or absence of NMDA (0-200 [mu]m).

Results: Volatile anesthetics reduced excitotoxin induced lactate dehydrogenase release by up to 52% in a dose-dependent manner. At higher concentrations, this protection was reversed. When corrected for olive oil solubility, the three anesthetics offered equivalent protection. MK-801 provided near-complete protection. Ca++ uptake was proportionally reduced with increasing concentrations of anesthetic but did not account for reversal of protection at higher anesthetic concentrations. Given equivalent NMDA-induced Ca++ loads, cells treated with volatile anesthetic had greater lactate dehydrogenase release than those left untreated. At protective concentrations, volatile anesthetics partially inhibited NMDA-induced mitochondrial membrane depolarization. At higher concentrations, volatile anesthetics alone were sufficient to induce mitochondrial depolarization.     

肠缺血再灌流时肠内给予葡萄糖能增加肠粘膜血流量

目的探讨肠缺血再灌流损伤时肠内营养与肠粘膜血流改变的关系.方法SD大鼠分为三组,先制作空肠袋,然后将激光多谱勒探头和肠粘膜张力计放置在空肠袋两端,分别向袋内注射丙氨酸、葡萄糖及甘露醇.用动脉夹阻断肠系膜上动脉血流60分后,再恢复灌流60分.分别测定肠粘膜血流量和局部PCO2张力(PrCO2).结果缺血再灌流过程中,与甘露醇组比较,葡萄糖组粘膜血流量显著增加,PrCO2降低.结论缺血再灌流过程中,肠内给予葡萄糖能增加肠粘膜血流量,对缺血再灌流损伤的肠道提供保护作用.     

Heart 7-hydroperoxycholesterol and oxysterols are elevated in chronically ethanol-fed rats.

Recently, cholesterol hydroperoxides have been shown to be sensitive pathogenic markers of reactive oxygen species (ROS)-mediated damage though they have never been measured in heart tissue. We hypothesized that cholesterol hydroperoxides and oxysterols, putative cardiotoxic products of cholesterol oxidation, are elevated in the hearts of alcoholics as a consequence of ROS-mediated reactions. To test this, we measured 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ol (7alpha-OOH and 7beta-OOH) by HPLC with postcolumn chemiluminescence as well as 7alpha- and 7beta-hydroxycholesterol (7alpha-OH and 7beta-OH) and 3beta-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto) by HPLC-UV in cardiac muscle of alcohol-fed rats. Alcohol feeding was carried out using a pair-feeding protocol with 35% of total dietary energy as ethanol; controls were pair-fed isocaloric glucose. After 6-7 wk treatment with alcohol, heart 7alpha-OOH, 7beta-OOH and 7beta-OH were significantly greater than in controls. Levels of heart phospholipid 16:0 and 18:1 were lower than in controls, while 18:0 and 18:2 were greater. This is the first report of the presence of 7alpha-OOH, 7beta-OOH and 7alpha-OH in cardiac tissue. The elevations in 7alpha-OOH and 7beta-OOH as well as 7beta-OH are evidence of increased oxidative stress and possible membrane changes. Alterations in the proportions of 16:0, 18:1, 18:2 and 18:0 in heart phospholipids provide further evidence of an altered membrane domain.     

Novel mutation p.Gly59Arg in GJB6 encoding connexin 30 underlies palmoplantar keratoderma with pseudoainhum, knuckle pads and hearing loss

Background  Connexins, components of the gap junction, are expressed in several organs including the skin and the cochlea. Mutations in connexin genes including GJB2 (Cx26), GJB3 (Cx31), GJB4 (Cx30.3), GJB6 (Cx30) and GJA1 (Cx43) are responsible for various dermatological syndromes and/or inherited hearing loss, frequently showing overlapping phenotypes.
Objectives  To clarify the spectrum of clinical phenotypes caused by connexin mutations.
Methods  We report a 32-year-old Japanese woman with mild palmoplantar keratoderma (PPK) with severe sensorineural hearing loss, knuckle pads and pseudoainhum of her toes.
Results  Direct sequencing revealed no mutation in GJB2 , but a novel heterozygous missense mutation p.Gly59Arg in GJB6 . Electron microscopy revealed no apparent morphological abnormality of gap junctions in the patient's lesional epidermis.
Conclusions  The patient harboured the novel GJB6 missense mutation p.Gly59Arg in the first extracellular loop of Cx30. Mutations in glycine 59 of Cx26 are associated with PPK–deafness syndrome, and the similar phenotype here supports the observed heteromeric channel formation; the dominant nature of the mutation suggests an effect on gap junctions similar to that of the comparable mutation in Cx26.     

Cytosolic acetyl-CoA synthetase affected tumor cell survival under hypoxia: the possible function in tumor acetyl-CoA/acetate metabolism

Understanding tumor-specific metabolism under hypoxia is important to find novel targets for antitumor drug design. Here we found that tumor cells expressed higher levels of cytosolic acetyl-CoA synthetase (ACSS2) under hypoxia than normoxia. Knockdown of ACSS2 by RNA interference (RNAi) in tumor cells enhanced tumor cell death under long-term hypoxia in vitro . Our data also demonstrated that the ACSS2 suppression slowed tumor growth in vivo . These findings showed that ACSS2 plays a significant role in tumor cell survival under hypoxia and that ACSS2 would be a potential target for tumor treatment. Furthermore, we found that tumor cells excreted acetate and the quantity increased under hypoxia: the pattern of acetate excretion followed the expression pattern of ACSS2. Additionally, the ACSS2 knockdown led to a corresponding reduction in the acetate excretion in tumor cells. These results mean that ACSS2 can conduct the reverse reaction from acetyl-CoA to acetate in tumor cells, which indicates that ACSS2 is a bi-directional enzyme in tumor cells and that ACSS2 might play a buffering role in tumor acetyl-CoA/acetate metabolism. ( Cancer Sci 2009; 100: 821–827)     

Epidermal growth factor receptor lacking C-terminal autophosphorylation sites retains signal transduction and high sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor

Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746_A750 EGFR; delE746_A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746_A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR. ( Cancer Sci 2009; 100: 552–557)