The surfaces of core histones in nucleosome are exposed as required for factor recognition, or buried for histone-DNA and histone-histone interactions. To understand the mechanisms by which nucleosome structure and function are coordinately altered in DNA-mediated reactions, it is essential to define the roles of both exposed and buried residues and their functional relationships. For this purpose, we developed GLASP (GLobal Analysis of Surfaces by Point mutation) and GLAMP (GLobal Analysis of Mutual interaction surfaces of multisubunit protein complex by Point mutation) strategies, both of which are comprehensive analyses by point mutagenesis of exposed and buried residues in nucleosome, respectively. Four distinct DNA-mediated reactions evaluated by Ty suppression (the Spt ) phenotype), and sensitivities to 6-azauracil (6AU), hydroxyurea (HU), and methyl methanesulfonate (MMS), require common and different GLAMP residues. Mutated GLAMP residues at the interface between histones H2A and H2B mainly affect the Spt ) phenotype but not HU and MMS sensitivities. Interestingly, among the mutated GLAMP residues surrounding the histone H3-H3¢ interface, some equally affect the Spt ) phenotype, and HU and MMS sensitivities, whereas others differentially affect the Spt ) phenotype, and HU and MMS sensitivities. Based on these and other results, the functional relationships among chromatin factors and GLASP and GLAMP residues provide insights into nucleosome disassembly ⁄ assembly processes in DNA-mediated reactions.

(R)-Thalidomide was oxidized to 5-hydroxythalidomide and 5’-hydroxythalidomide by NADPH-fortified liver microsomes from humans and monkeys. (R)-Thalidomide was hydroxylated more efficiently than (S)-thalidomide. Recombinant human P450s 3A4, 3A5, and 3A7 and monkey P450s 3A8 and 3A5 (co-expressed with NADPH-P450 reductase in bacterial membranes) also catalyzed (R)-thalidomide 5-hydroxylation. Purified human P450s 2C19, 3A4, and 3A5 mediated (R)-thalidomide 5-hydroxylation at similar rates in reconstituted systems. P450 2C19 showed a rather non-saturable substrate-velocity curve; however, P450s 3A4 and 3A5 showed sigmoidal curves. P450 also oxidized 5-hydroxythalidomide to an epoxide or dihydroxy compound. Liquid chromatography-mass spectrometry analysis revealed formation of a glutathione conjugate from (R)- and (S)-5-hydroxythalidomide, catalyzed by liver microsomal P450s 3A4 and 3A5 in the presence of glutathione (assigned as a conjugate of 5-hydroxythalidomide formed on the phenyl ring). These results indicate that human P450s 3A4 and 3A5 mediate thalidomide 5-hydroxylation and further oxidation leading to a glutathione conjugate, which may be of relevance in the pharmacological and toxicological actions of thalidomide.

Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 alpha-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli-C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5'-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert L-lysine to cadaverine, as there was no accumulation of L-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources.

ABSTRACT:There is growing clinical interest of thalidomide because of its immunomodulatory and antiangiogenic properties, despite its teratogenicity. However, little information about thalidomide has been reported regarding its precise effects on drug-metabolizing enzymes. We investigated the effects of thalidomide on cytochrome P450 (P450) enzymes in human liver microsomes to clarify the potential for possible drug interactions. Thalidomide inhibited S-mephenytoin 4-hydroxylation activities of recombinant P450 2C19 and human liver microsomes: the apparent concentration of thalidomide producing 50% inhibition was approximately 270 M for P450 2C19. Midazolam 4-hydroxylation activities were suppressed by thalidomide, but activities of 1-hydroxylation and total midazolam oxidation and testosterone 6␤-hydroxylation were enhanced in the presence of thalidomide. Recombinant P450 3A5 was found to have altered kinetics at clinically relevant concentrations of thalidomide (10-30 M). P450 3A4 was also affected, but only at higher thalidomide concentrations. Enhanced midazolam hydroxylation by thalidomide was also seen in liver microsomal samples harboring the CYP3A5*1 allele. Similarly enhanced rates of cyclosporine A clearance were observed in P450 3A5 and liver microsomes expressing P450 3A5 in the presence of thalidomide. A proposed effector constant for thalidomide corresponded roughly to its clinical plasma levels. Docking studies with a P450 3A5 homology model, based on the published structure of P450 3A4, revealed close interaction between thalidomide and the heme of P450 3A5. The present results suggest that total midazolam metabolism or cyclosporine A clearance may be increased by thalidomide in a dose-dependent manner. Unexpected drug interactions involving thalidomide might occur via heterotropic cooperativity of polymorphic P450 3A5.

No abstract

Understanding changes in excited state properties under the influence of an external stimulus, such as pressure or temperature, is important in the context of optimizing molecular components for a number of applications including sensors and imaging reagents. Herein, we use UV/Vis absorption, fluorescence and excitation spectroscopies and fluorescence lifetime measurements supported by calculations to probe the effect of hydrostatic pressure on the excited state characteristics of a conformationally-divergent mechanochromic compound PTZ-DBPHZ (containing two phenothiazine moieties attached to a dibenzo[a,j]phenazine core) in toluene and methylcyclohexane. We demonstrate that hydrostatic pressure can be used to manipulate the equilibria between excited state conformers. This work provides new perspectives for mechanoresponsive materials and as an attractive alternative to conventional ratiometric sensors.[a] Prof.

We devised a three-dimensional method for estimation of cerebral development and myelination which measures cerebral volume using MRI. Accuracy of the system was estimated using cadaver brains. The mean percentage error in the calculated volumes compared with the real volumes was 2.33%, range 0.00-5.33%. We applied the method to the volume of both cerebral hemispheres (CH), basal ganglia, thalamus and internal capsule (BT), and myelinated white matter (WM) in 44 neurologically normal individuals (4 months to 28 years of age), 13 patients with spastic motor disturbances (2-25 years of age), and 9 patients with athetotic motor disturbances (2-23 years of age). In the neurologically normal cases, the volumes of CH, BT and WM increased with age; the volume of MW more slowly than that of CH. In cases with spastic motor disturbances, the volumes of CH, BT and WM were between -1.4 and 3.5 SD, -1.0 and -3.5 SD, and 0.0 and -5.2 SD respectively, of those of neurologically-normal cases. On the other hand, 7 of the 9 cases with athetotic motor disturbances were within 2 SD of the volume of CH in neurologically normal cases. Our method for direct measurement of cerebral volume based on serial MRI should be useful for the accurate assessment of brain development and quantitative analysis of delayed myelination.

in enhancing coping skills, increasing social support, and reducing stress responses. Stress management intervention studies in the workplace have increased during the past two decades [1][2][3][4][5][6][7][8][9][10][11][12][13] . It is often assumed that there are two basic approaches to interventions in the workplace 1,9,10) . One is an organization-oriented approach, which aims to identify and improve a stressful work environment, and the other is individual-oriented and aims to enhance the coping abilities of individual employees. The latter approach is of particular interest to occupational health psychologists, who regard employee stress responses as the results of coping with stressful work environments, and who frequently help employees cope more effectively 11) . Our study was conducted on the basis of the individualoriented approach.There are various kinds of intervention techniques in the individual-oriented approach. Cognitive-behavioral training, stress measurement and personalized feedback, meditation, relaxation training, and physical fitness training are all examples. Accumulated experiences from individual-oriented intervention showed that cognitivebehavioral training or a combination of cognitivebehavioral training with relaxation training was more effective than other techniques 11,12) . In accord with that evidence, our intervention program was planned combining the two techniques.All participants in this study were teachers, for whom job stress has become a problem of especially serious proportions because it affects on their psychological wellbeing and the future of the profession. In Japan, 0.24% of teachers were suspended from their jobs in 2000 because of a mental disorder; the highest rate for the last University Graduate School of Education-The aim of this study was to examine the effects of a stress management program for teachers on their stress responses, social support, and coping. Participants (n=24) were assigned to either an intervention or a waiting list control group. A five-session program, including psychoeducation, group discussion, roleplaying and relaxation training, was conducted for the intervention group at two week intervals. Eight participants from each of the groups responded to preand post-intervention questionnaire surveys. The positive intervention effect was significant for social support from co-workers (p=0.035), whereas the negative intervention effect was significant for proactive coping (p=0.033). No significant effect was observed for stress responses (vigor, anger, fatigue, anxiety, depression, and somatic stress responses) (p>0.05). The positive intervention effect was marginally significant for social support from co-workers (p=0.085) and anger (p=0.057) among those who at first had high stress response scores in the pre-intervention survey (n=5 and n=4 for the intervention and waiting list control groups, respectively). Furthermore, the positive intervention effect was significant for social support from co-workers (p=0.021) and marginally significant ...