Sequence specificity in the interaction of the four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides with the supF gene

The shuttle vector pS189 was treated with each of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide, and the modified DNA was used as a template in a polymerase arrest assay examining the supF gene. Sites at which polymerase (Sequenase, version 2.0) progress along the template was blocked were presumed to be at or near sites of adduct formation. The polymerase arrest sites were compared with recently reported mutation hotspots induced by these agents in this gene (Bigger et al., Proc. Natl. Acad. Sci. USA, 89: 368-372, 1992). For 31 of 32 mutation hotspots, a polymerase arrest band was present at or 1 or 2 nucleotides 3'- to that site, indicating that adduct formation tended to be associated with mutation hotspots. However, the arrest bands near mutation hotspots were not particularly prominent in all cases, and there were many sites of substantial polymerase arrest that were not in the vicinity of mutation hotspots. Thus, factors in addition to chemical selectivity must play key roles in determining sites of mutation.     

Analysis of c-Ki-ras oncogene and p53 immunocytochemistry in the gallbladder mucosa of an experimental dog model of anomalous arrangement of the pancreaticobiliary ducts

Anomalous arrangement of the pancreaticobiliary ducts is a congenital condition which predisposes the affected person to biliary tract carcinoma. We developed an experimental dog model of anomalous arrangement of the pancreaticobiliary ducts to investigate the mechanism of carcinogenesis in this condition. We used this model to analyze point mutations in the c-Ki-ras gene, and to assess the expression of mutant p53 protein in the gallbladder mucosa. The histopathological appearance of the gallbladder mucosa was also examined. Glandular structures were seen in four of seven (57%) gallbladders examined 14 months after the surgical creation of an anastomosis between the gallbladder and the pancreatic duct. Goblet cells were seen in two of seven gallbladders (29%). However, dot-blot hybridization and immunohistochemical study did not reveal any mutations in the c-Ki-ras gene, or any over-expression of the p53 protein in the specimens. These results show that the gallbladder mucosa is damaged by refluxing pancreatic juice in this dog model of anomalous arrangement of the pancreaticobiliary ducts, but that severe damage may be necessary to induce mutations in the c-Ki-ras proto-oncogene, or in the p53 gene.     

Characteristics of Escherichia coli HB101 and Pseudomonas putida PpY101 harboring a recombinant plasmid with tandem insertion of the mercury resistance operon

We constructed the plasmid pSUPmer2 by inserting tandem copies of the mercury resistance (mer) operon into a broad host range-vector, and introduced it into Escherichia coli HB101 and Pseudomonas putida PpY101 to increase their mercury resistance. Strains harboring plasmid pSUPmer2 had higher mercury resistance and mercuric reductase activity than those strains harboring the plasmid pSUPmer which had one copy of the mer operon. Mercury resistance of P. putida PpY101 was significantly increased by tandem insertion of the mer operon.     

Anomalous wavelength changes of 980-nm pump laser diodes with antireflection coatings

We have observed abrupt wavelength jumps form 980-nm pump laser diodes with an antireflection (AR) coating when the injection currents were changed. The width of the anomalous jumps becomes wider as the cavity length is shortened. It was clarified from theoretical calculations that the phenomenon is caused by narrow mirror loss distributions. It was also confirmed that wide AR coatings or a long cavity can avoid these abrupt wavelength jumps.     

Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1-ATPase beta subunit determined by 13C-NMR

Glutamic acid-190 in the beta subunit of F1-ATPase from thermophilic Bacillus PS-3 (TF1) was reported to be essential for the ATPase activity. The mutant TF1beta subunit in which Glu-190 had been substituted by cysteine was carboxymethylated with 13C-labeled monoiodoacetic acid. The pKa value of the carboxymethylene group at the 190 position was determined as 5.6 +/- 0.4 by 13C-NMR. On the basis of this value, the pKa of the carboxylate of Glu-190 of the TF1beta subunit was estimated to be 6.8 +/- 0.5. The unusually high pKa could play a role in the catalytic mechanism of F1-ATPase.     

Catalytic properties of adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki

Adenylylsulfate reductase (EC 1.8.99.2) isolated from Desulfovibrio vulgaris Miyazaki catalyzes electron transfer from dihydroflavin coenzyme (FADH2, FMNH2, or dihydroriboflavin) to adenylyl sulfate (APS), and catalyzes flavin-mediated oxidation of ferrocytochrome c3 with APS. The reaction with FAD as an electron mediator was markedly stimulated in the presence of menadione. Km of the enzyme was about 0.015 mM for riboflavin and FAD in the presence of menadione. Free flavin coenzyme was found to be the normal cellular constituent. These observations suggested that free flavin coenzyme may be a physiological electron carrier for APS reductase, and the enzyme may be called AMP, sulfite:flavin oxidoreductase. Km (APS) of this enzyme is lower than 1 microM. The enzyme is not inhibited by ATP and GTP, but was inhibited by AMP and sulfite. Its extremely low Km (APS) enables this enzyme to reduce any traces of cytosolic APS which is present only at micromolar concentration, and inhibition by sulfite makes this organism utilize an energetically favorable electron acceptor, sulfite, preferentially over APS which is produced from sulfate at the cost of ATP.     

Distinct mechanisms for expression of Fos-like immunoreactivity and synaptic potentiation in telencephalic hyperstriatum of the quail chick

In the intermediate and medial hyperstriatum ventrale (IMHV), a telencephalic region essentially involved in the initial processes of early learning tasks in poultry chicks, induction of an immediate early gene c-fos correlates significantly with the degree of learning (K.V. Anokhin, R. Mileusnic, I.Y. Shamakina, S.P.R. Rose, Effects of early experience on c-fos gene expression in the chick forebrain, Brain Res. 544 (1991) 101-107; B.J. McCabe, G. Horn, Learning-related changes in Fos-like immunoreactivity in the chick forebrain after imprinting, Proc. Natl. Acad. Sci. USA 91 (1994) 11417-11421). In slices of IMHV in vitro, on the other hand, tetanic stimulation at a low frequency induces a potentiation of synaptic responses (P.M. Bradley, B.D. Burns, A.C. Webb, Potentiation of synaptic responses in slices from the chick forebrain, Proc. R. Soc. Lond. B. 243 (1991) 19-24; T. Matsushima, K. Aoki, Potentiation and depotentiation of DNQX-sensitive fast excitatory synaptic transmission in telencephalon of the quail chick, Neurosci. Lett. 185 (1995) 179-182). In this study, we have examined a possible causal link between these two forms of activity-dependent processes, c-fos expression and synaptic potentiation. C-fos was visualized immunohistochemically using antibody raised against the Fos-protein, and potentiation was evaluated on the basis of field potential responses to local electrical stimulation. Tetanic stimulation (5 Hz x 300 pulses) was required for potentiation, but not for c-fos expression. Conversely, a negative correlation appeared between them, and slices with relatively high density of Fos-like immunoreactive cells around the stimulation site failed to show potentiation. Furthermore, drugs similarly effective in blocking potentiation (such as AP5 (NMDA receptor antagonist) and bicuculline (GABA(A) receptor antagonist)) had different effects on the c-fos induction. While AP5 had minor, if any, effects on c-fos expression, bicuculline enhanced it selectively around the site of stimulation. Our results suggest that these two processes are basically distinct, and could represent different aspects in the formation of memory traces in IMHV.     

LIFE PREDICTION OF 316 STAINLESS STEEL UNDER CREEP-FATIGUE LOADING

Life prediction for creep-fatigue loading conditions should be related to creep damage mechanisms. In order to examine the effect of the creep damage mode on rupture life under creep-fatigue loading, a “combined creep-fatigue loading test” was carried out on 316 stainless steel. In this method, creep loading and fatigue loading are repeated alternately. The fracture criteria under combined loading closely depend on the creep fracture modes of the static creep test. A new life prediction method which uses this new fracture criterion is proposed. The criteria are changed when the creep damage mode varies. In order to verify the adequacy of this method, fatigue tests with a tensile strain-hold wave form were carried out. It is clear that rupture life in such fatigue tests is dependent on the chosen fracture criteria.     

Low superoxide dismutase activity in schizophrenic patients with tardive dyskinesia

BACKGROUND: Tardive dyskinesia (TD) is a therapy-resistant adverse effect of neuroleptics. Although the exact pathophysiology of TD is unknown, oxygen radicals have been speculated to play a role in TD based on several lines of evidence. Superoxide dismutase (SOD) is a key enzyme which scavenges oxygen radicals. The authors investigated the association between erythrocyte SOD activity and TD. METHODS: Erythrocyte SOD activities were measured, blinded as to the presence or absence of TD. In 30 patients with schizophrenia who had been on typical neuroleptics for more than 10 years. TD severity was independently assessed, using the abnormal involuntary movement scale (AIMS), by two raters. RESULTS: There was a significant decrease in erythrocyte SOD activity in the definite TD group (N = 10) as compared with the no TD (N = 8) and questionable TD (N = 12) groups. Erythrocyte Cu,Zn-SOD activities correlated with AIMS scores. CONCLUSIONS: Patients with TD had low SOD activities as compared to those without TD. As a causal link between SOD activity and TD was not established in this study, larger prospective studies are warranted to determine whether patients with low SOD activity are susceptible to neuroleptic-induced TD.     

Cross-bridge movement in rat slow skeletal muscle as a function of calcium concentration

A single fibre bundle from rat soleus muscle was chemically skinned with saponin and the transfer of myosin heads from the thick filaments to the thin filaments at a sarcomere length of 2.4 microm was measured as a function of Ca2+ concentration using an x-ray diffraction method at 4-7 degrees C. In the relaxed state, the 1,0 spacing was 42.08 nm. The spacing showed no significant decrease when the Ca2+ concentration was below the threshold (-log10 [Ca2+] or pCa 5.8). No significant transfer of the myosin heads occurred when the Ca2+concentration was below the threshold (pCa 5.8). When the muscle was maximally activated at pCa 4.4, the spacing decreased to 40.35 nm. During the maximum isometric contraction at pCa 4.4, 54. 9 +/- 6.5% (+/-SE of the mean) of the myosin heads were transferred to the thin filaments. The transfer of the myosin heads was approximately proportional to relative tension. These results suggest that myosin heads of both fast-twitch and slow-twitch skeletal muscles transferred on the common movement as a function of Ca2+ concentration.