Interaction of SeqA and Dam methylase on the hemimethylated origin of Escherichia coli chromosomal DNA replication

Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam methylase. Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end of oriC were examined. We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers. Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R. On the other hand, Dam methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences. The binding specificity and higher affinity of SeqA protein over Dam methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam methylase to the newly replicated oriC for the sequestration of chromosomal replication. Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam methylase, and most SeqA protein spontaneously dissociated 10 min after binding. Also, SeqA protein delayed the in vitro methylation of hemimethylated 13-mers by Dam methylase. These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.     

The p67(phox) activation domain regulates electron flow from NADPH to flavin in flavocytochrome b(558).

An activation domain in p67(phox) (residues within 199-210) is essential for cytochrome b(558)-dependent activation of NADPH superoxide (O2(-.)) generation in a cell-free system (Han, C.-H., Freeman, J. L. R., Lee, T., Motalebi, S. A., and Lambeth, J. D. (1998) J. Biol. Chem. 273, 16663-16668). To determine the steady state reduction flavin in the presence of highly absorbing hemes, 8-nor-8-S-thioacetamido-FAD ("thioacetamido-FAD") was reconstituted into the flavocytochrome, and the fluorescence of its oxidized form was monitored. Thioacetamido-FAD-reconstituted cytochrome showed lower activity (7% versus 100%) and increased steady state flavin reduction (28 versus <5%) compared with the enzyme reconstituted with native FAD. Omission of p67(phox) decreased the percent steady state reduction of the flavin to 4%, but omission of p47(phox) had little effect. The activation domain on p67(phox) was critical for regulating flavin reduction, since mutations in this region that decreased O2(-.) generation also decreased the steady state reduction of flavin. Thus, the activation domain on p67(phox) regulates the reductive half-reaction for FAD. This reaction is comprised of the binding of NADPH followed by hydride transfer to the flavin. Kinetic deuterium isotope effects along with K(m) values permitted calculation of the K(d) for NADPH. (R)-NADPD but not (S)-NADPD showed kinetic deuterium isotope effects on V and V/K of about 1.9 and 1.5, respectively, demonstrating stereospecificity for the R hydride transfer. The calculated K(d) for NADPH was 40 microM in the presence of wild type p67(phox) and was approximately 55 microM using the weakly activating p67(phox)(V205A). Thus, the activation domain of p67(phox) regulates the reduction of FAD but has only a small effect on NADPH binding, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.     

Muscarinic cholinergic signaling in cardiac myocytes: dynamic targeting of M2AChR to sarcolemmal caveolae and eNOS activation

The isoform of nitric oxide synthase (eNOS or NOS3) originally described in endothelial cells is also expressed in a number of other cell types, including cardiac myocytes. eNOS is activated in both atrial and ventricular myocytes, including specialized pacemaker cells, by M2AChR agonists, among other stimuli. In cardiac myocytes, as in endothelial cells, eNOS is targeted to sarcolemmal caveolae, due to both co-translational myristoylation and later palmitoylation, and by the presence of a caveolin binding domain in eNOS which interacts with the caveolin scaffolding domain. In the absence of ligand, the M2AChR is not associated with caveolar microdomains, but translates into caveolae upon agonist (but not antagonist) binding. Finally, the role of M2AChR-induced eNOS activation in regulating I(Ca-L) via activation of guanylyl cyclase has been confirmed in ventricular myocytes of mice that lack functional eNOS (i.e., eNOS(null)).     

The inverted nipple: its grading and surgical correction

Inverted nipples have been treated by various methods by many authors, but the relationship between the grade of the deformity and the appropriate surgical procedure is not clearly described. One hundred seven inverted nipples in 60 patients were treated from 1993 to 1997. They were divided into three groups by the authors' system of grading. The grade was made by preoperative evaluation of severity of inversion and was confirmed by the surgical findings. In grade I, the nipple is easily pulled out manually and maintains its projection quite well. Grade I nipples are believed to have minimal fibrosis; thus, manual traction and a single, buried purse-string suture are enough for the correction. The majority of inverted nipples belong to grade II, i.e., the nipples can be pulled out but cannot maintain projection and tend to go back again. These nipples are thought to have moderate fibrosis beneath the nipple. Blunt dissections for surgical release were carried out until the inversion did not recur after releasing the traction. The lactiferous ducts could be identified and preserved, permitting proper release of fibrotic bands in the grade II group. The purse-string suture was used. In grade III, to which the least number of inverted-nipple cases belong, the nipple can hardly be pulled out manually. Severe fibrosis made it impossible to reach optimal release of the fibrotic band with the preservation of the ducts. The fibrotic bands are widely dissected, and the lactiferous ducts are cut, especially in the central portion. Two or three deepithelialized dermal flaps may be used to make up for soft-tissue deficiency; a purse-string suture is also used. This grading system will be useful for patient classification and analysis, systematic planning, and application of the proper surgical procedures.     

The chemopreventive effects of tea on human oral precancerous mucosa lesions

A double-blind intervention trial was conducted in patients with oral mucosa leukoplakia using a mixed tea product developed by the authors. Fifty-nine oral mucosa leukoplakia patients, diagnosed by established clinical and pathological criteria, were randomly divided into a treated group (3 g mixed tea oral administration and topical treatment) and a control group (placebo and glycerin treatment). After the 6-month trial, the size of oral lesion was decreased in 37.9% of the 29 treated patients and increased in 3.4%; whereas the oral lesion was decreased in 10.0% of the 30 control patients and increased in 6.7%. At the same time, the incidence of micronucleated exfoliated oral mucosa cells in the treated group (5. 4 per 1000 cells) was lower than that in the control group (11.3 per 1000 cells)(P < 0.01); whereas it was 1.4 per 1000 cells in 20 healthy subjects. The micronuclei and chromosome aberration rate in the peripheral blood lymphocytes showed the same results. In pathological examination, there were significant differences (P < 0. 05) in the number and total volume of the silver-stained Nucleolar Organizer Regions (AgNOR) and the proliferating index of Proliferation Cell Nuclear Antigen (PCNA) in oral mucosa cell nuclei between the treated group and the control group which indicates that cell proliferation was decreased in the treated patients. The overall results provide some direct evidence on the protective effects of tea on oral cancer.