Measurement of O-6-methylguanine-DNA methyltransferase activity using oligonucleotides and restriction enzyme in human brain tumors

We determined the levels of MGMT activity in 46 human brain tumors, using oligonucleotides containing O-6-methylguanine at a PvuII recognition site, and correlated MGMT activity with chemotherapeutic effectiveness in 14 patients with high-grade gliomas who received ACNU chemotherapy. The results characterize MGMT activities among different types and within individual types of human brain tumors. A certain fraction of gliomas exhibited a low value of MGMT activity. Tumors showing a low MGMT activity responded well to ACNU and resulted in prolongation in the time to tumor progression. Thus, the low MGMT activity of gliomas may provide a theoretical basis for application of CENU chemotherapy.     

Lidocaine-metabolizing Activity after Warm Ischemia and Reperfusion of the Rat Liver In Vivo

The effect of warm ischemia on lidocaine-metabolizing activity was examined in vivo. Total liver ischemia was produced for 1 hr in Sprague-Dawley rats by clamping the portal vein and hepatic artery at the hilum. Livers were then reperfused, and liver microsomes were prepared before and 0, 2, 6, and 24 hr, and 3, 6, and 10 days after reperfusion. Microsomal lidocaine-metabolizing activity and cytochrome P-450 content were examined. Lidocaine N-deethylase activity was decreased from 2.25 ± 0.33 to 0.97 ± 0.21 nmol/mg protein/min (mean ± SD) 24 hr after reperfusion. This inhibition was prolonged, and activity gradually recovered after 10 days. The cytochrome P-450 content showed the same tendency. On the other hand, serum levels of alanine aminotransferase increased significantly 2 hr after reperfusion and returned to control levels 3 days after reperfusion. Liver blood flow recovered rapidly after unclamping and reached baseline levels within 6 hr. Our results suggest that after warm ischemia, prolonged hepatic dysfunction in drug metabolism, which cannot be detected by evaluating serum enzymes or liver blood flow, exists at the microsomal level.     

Bone marrow stromal-B cell interactions in polycyclic aromatic hydrocarbon-induced pro/pre-B cell apoptosis.

Environmental polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons are immunotoxic in a variety of systems. In a model system of B lymphopoiesis, PAH exposure rapidly induces apoptosis in CD43- pre-B and CD43+ pro/pre-B cells. Apoptosis induction by 7,12-dimethylbenzo[a]anthracene (DMBA) is dependent upon AhR+ bone marrow stromal cells and likely involves DMBA metabolism within the stromal cell. However, it is not known if PAH-treated stromal cells release free metabolites or soluble factors that may directly induce B cell death or if the effector death signal is delivered by stromal cell-B cell contact. Here, we demonstrate that supernatants from DMBA-treated bone marrow stromal cells contain an activity capable of inducing apoptosis in pro/pre-B cells cocultured with stromal cells. This activity (1) is not produced when stromal cells are cotreated with DMBA and alpha-naphthoflavone (alpha-NF), an aryl hydrocarbon receptor (AhR) and cytochrome P-450 inhibitor, (2) is > or = 50 kDa, (3) is trypsin and heat sensitive, and (4) is dependent on AhR+ stromal cells, which in turn deliver the effector death signal to pro/pre-B cells. The results (1) argue against a role for a soluble, stromal cell-derived cytokine as the effector of PAH-induced pro/pre-B cell death, (2) exclude the possibility of a free metabolite acting directly on AhR- pro/pre-B cell targets, and (3) suggest the elaboration by stromal cells of a relatively stable, DMBA metabolite-protein complex capable of acting on other stromal cells at some distance. Collectively, these studies suggest that, while stromal cell products, e.g., metabolite-protein complexes, may affect the function of distant stromal cells, the effector death signal delivered by stromal cells to bone marrow B cells is mediated by cell-cell contact.     

Prognostic significance of c-kit mutation in localized gastrointestinal stromal tumors.

PURPOSE: Constitutive mutational activation of c-kit has been found to be associated with the pathogenesis of gastrointestinal stromal tumors (GISTs). The prognostic significance of c-kit mutations, however, is still controversial. EXPERIMENTAL DESIGN: We examined 86 patients curatively resected for localized GIST. Genomic DNA was extracted from paraffin-embedded tumor tissues. Exons 9, 11, 13, and 17 of the c-kit gene were amplified by PCR and sequenced. RESULTS: Mutations in exon 11 were detected in 61 tumors, and mutations in exon 9 were observed in three tumors, whereas no mutations were detected in exons 13 or 17. The overall c-kit mutation frequency was 74%. Amino acid alterations in the 61 tumors with exon 11 mutations were deletion in 33 tumors, substitution in 20, both deletion and substitution in 4, insertion in 1, and duplication in 3. Histologically, tumors with c-kit mutations showed higher mitotic counts and higher cellularity. The 5-year relapse-free survival (RFS) in patients having GISTs with c-kit mutations was 21%, compared with 60% in those without c-kit mutations. Significantly higher RFS rates were observed in patients with tumors having mitotic counts < 5 mitoses/50 high power field, spindle-cell histology, tumor size < 5 cm, or gastric GISTs. Multivariate analyses indicated association of poorer RFS with a higher mitotic count > or = 5 of 50 high power fields; odds ratio (OR) = 3.0], presence of c-kit mutations (OR = 5.6), and a larger tumor size (> or = 5 cm; OR = 4.2). CONCLUSIONS: The presence of c-kit mutation, along with high mitotic count and larger tumor size, was an independent factor for poor prognosis in patients with localized GISTs.     

Fas-Fas ligand system in the peripheral blood of patients with renal diseases

To clarify the role of the Fas-Fas ligand (FasL) system in the peripheral blood from patients with various renal diseases, the Fas and FasL expression on mononuclear cells (MNCs) and serum levels of soluble Fas (sFas) and soluble FasL were investigated. Patients were selected from those with various types of glomerular diseases showing various degrees of renal function. Fas expression on MNCs was analyzed by a FACScan, sFas and soluble FasL were measured with an ELISA kit, and FasL expression on MNCs was counted using a FACScan after a bioassay. Fas-positive MNCs and sFas increased with statistical significance concomitantly with deterioration in renal function. Moreover, there was a significant correlation between them. sFas- and FasL-positive MNCs were significantly correlated with proteinuria. However, the Fas expression percentage on MNCs and/or serum levels of sFas did not correlate with the number of TUNEL-positive cells in the glomeruli. Also, there was no disease specificity in the activation of Fas. These results indicate that Fas expression on MNCs is activated in accordance with the deterioration in renal function without disease specificity, corresponding to the elevation of serum sFas levels to protect against Fas-mediated apoptosis.     

Echocardiographic assessment of patent ductus arteriosus shunt flow pattern in premature infants

AIMS—To identify the patent ductus arteriosus (PDA) shunt flow pattern using Doppler echocardiography; and to assess whether it could be used to predict the development of clinically significant PDA.
METHODS—Premature infants weighing under 1500 g, who required mechanical ventilation, and in whom daily echocardiography could be performed from day 1 until the ductus closed, and on day 7 to confirm closure, were studied. The PDA shunt flow was identified from four Doppler patterns, and the closed pattern of a closed duct was also presented. Clinically significant PDA was diagnosed when there was colour Doppler echocardiographic evidence of left to right ductal shunt associated with at least two of the following clinical signs: heart murmur (systolic or continuous); persistent tachycardia (heart rate>160/min); hyperactive precordial pulsation; bounding pulses; and radiographic evidence of cardiomegaly or pulmonary congestion.
RESULTS—Of 68 infants enrolled into this study, clincally significant PDA developed in 31. The most recordable sequence of transition change of shunt flow pattern for clinically significant PDA was: pulmonary hypertension pattern, to growing pattern, to pulsatile pattern, to closing pattern, to closed pattern. And that for non-clinically significant PDA was: pulmonary hypertension pattern, to closing pattern, to closed pattern. The growing and the pulsatile patterns were mostly documented in infants with clinically significant PDA. The first documented growing pattern to predict clinically significant PDA gave a sensitivity of 64.5% and a specificity of 81.1%; the first documented pulsatile pattern gave a sensitivity of 93.5% and a specificity of 100%.
CONCLUSION—Doppler echocardiographic assessment of PDA shunt flow pattern during the first 4 days of life is useful for predicting the development of clinically significant PDA in premature infants. At that stage, the closing or closed Doppler pattern indicates that infants are not at risk of developing clinically significant PDA; the growing or pulsatile Doppler pattern indicates a continuing risk of developing clinically significant PDA.

     

Age-dependent change in the levels of Abeta40 and Abeta42 in cerebrospinal fluid from control subjects, and a decrease in the ratio of Abeta42 to Abeta40 level in cerebrospinal fluid from Alzheimer's disease patients

In order to address an age-dependent alteration in the concentration of beta-amyloid polypeptides (Abetas) within the central nervous system and its probable predisposition to amyloidgenesis in Alzheimer's disease (AD), we measured two species of soluble Abetas, Abeta40 and Abeta42, in cerebrospinal fluids (CSF) from randomly selected Japanese control subjects at various ages (n = 33) and then compared these data with those of probable Japanese AD patients (n = 23). CSF concentrations of Abeta40 and Abeta42 peptides were age-dependent (ANOVA, Bonferroni's multiple comparison; p < 0.01 and p < 0.05, respectively) and were lower in the infant than in adults. From mid-20, the Abeta40 concentrations were decreasing while Abeta42 were rather stable. Abetas in CSF from AD patients (n = 23), whose epsilon4 allele frequency of the apolipoprotein E gene was higher than in controls (n = 83, p < 0.03), were not statistically different from those of age-matched controls (n = 13). A linear relationship was detected between the Abeta40 concentration and the Mini-Mental State Examination score (p < 0.05). The ratio of the Abeta42 to the Abeta40 level measured in the AD CSF samples was approximately 38% decreased compared to age-matched controls (p < 0. 05). These data suggest that the physiological metabolism of soluble Abetas in the brain is regulated in an age-dependent manner, and that the ratio of Abeta42 to Abeta40 level in the CSF would be a useful marker for monitoring progression of AD.     

Age-dependent decline in the apolipoprotein E level in cerebrospinal fluid from control subjects and its increase in cerebrospinal fluid from patients with Alzheimer's disease

In order to address the significance of apolipoprotein E (apoE) in the pathogenesis as well as the clinical diagnosis of Alzheimer's disease (AD), we measured its level in cerebrospinal fluid (CSF) from randomly selected Japanese control subjects at various ages (n = 36), which included 14 age-matched controls, and from AD patients including early-onset (n = 11, EOAD) and late-onset (n = 14, LOAD) cases. The CSF apoE level in controls linearly decreased during aging to over 80 years (r(2) = 0.323, p < 0.0001). The CSF apoE level in AD patients was 31.9% elevated compared to the age-matched controls (n = 14, p < 0.05) and linearly increased with a decrement of the patients' Mini Mental State Examination scores. Moreover, the CSF apoE level of EOAD patients (n = 11) was higher than that of LOAD patients (n = 14, p < 0.05), whose APOE epsilon4 allele frequency was significantly higher than that of controls (chi(2) = 7. 16, p < 0.03). Two-dimensional gel electrophoretic analysis of the heparin-Mn(2+)-precipitable lipoprotein fraction in CSFs showed that the ratio between the level of CSF apoA-I and that of CSF apoE of controls was significantly higher than those of all AD and LOAD subjects (p < 0.01, p < 0.05), while the CSF apoA-I-to-apoE ratios of the two AD groups were not significantly different. These results suggest that overproduction of apoE protein may be a consequence of astroglial response to neurodegeneration in AD and that the determination of CSF apoliprotein levels serves as a clinical marker for monitoring the progression of AD.     

Thermosoftening treatment of the nasotracheal tube before intubation can reduce epistaxis and nasal damage

We evaluated whether a thermosoftening treatment with warm saline of a nasotracheal preformed tube can improve navigability through the nasal passageways and reduce epistaxis and nasal damage. A total of 150 patients were randomly allocated to three groups: Group I (untreated tube group, n = 50), Group II (35 degrees C treated tube group, n = 50), and Group III (45 degrees C treated tube group, n = 50). In Groups II and III, the tubes were softened at 35 +/- 2 degrees C and 45 +/- 2 degrees C with warm saline, respectively. In Group I the tube was prepared at room temperature (25 +/- 2 degrees C). The incidence of epistaxis and nasal damage in Groups II and III was significantly less than that of Group I (P: < 0.05). Despite the more frequent incidence of smooth passage in Group III, no statistical difference was found among the groups. Logistic regression analysis also confirmed that epistaxis was more likely to be reduced when the tube had been thermosoftened (odds ratio = 1.46, 95% confidence interval = 1.02, 2.11). We conclude that simple thermosoftening treatment of the nasotracheal tube with warm saline helps to reduce epistaxis and nasal damage. IMPLICATIONS: Thermosoftening treatment of a nasotracheal tube with warm saline before intubation can effectively reduce epistaxis and nasal damage. This technique is safe, easy, and suitable for all types of tubes and does not require additional implements.