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.     

A new locus for autosomal dominant stargardt-like disease maps to chromosome 4

Stargardt disease (STGD) is the most common hereditary macular dystrophy and is characterized by decreased central vision, atrophy of the macula and underlying retinal-pigment epithelium, and frequent presence of prominent flecks in the posterior pole of the retina. STGD is most commonly inherited as an autosomal recessive trait, but many families have been described in which features of the disease are transmitted in an autosomal dominant manner. A recessive locus has been identified on chromosome 1p (STGD1), and dominant loci have been mapped to both chromosome 13q (STGD2) and chromosome 6q (STGD3). In this study, we describe a kindred with an autosomal dominant Stargardt-like phenotype. A genomewide search demonstrated linkage to a locus on chromosome 4p, with a maximum LOD score of 5.12 at a recombination fraction of.00, for marker D4S403. Analysis of extended haplotypes localized the disease gene to an approximately 12-cM interval between loci D4S1582 and D4S2397. Therefore, this kindred establishes a new dominant Stargardt-like locus, STGD4.     

Time-resolved fluorescence immunoassay of thyroxine in serum: immobilized antigen approach

With T(4)-bovine IgG as a solid-phase antigen, we have developed a direct competitive-type immunoassay for serum total thyroxine (TT(4)), which depends on the competitive distribution of europium-labeled anti-T(4) monoclonal antibody between solid-phase-bound T(4) and the T(4) in the sample or standard. The captured fraction of the tracer was measured after a dissociation-enhancement step. Four different T(4) protein conjugates were synthesized, of which T(4)-bovine IgG was selected as the most favorable for the preparation of solid-phase antigen. The sensitivity was 3.5 ng/ml with a sample volume of 20 microl. T(4) values obtained by this procedure agreed well with those obtained by RIA (r = 0.967, n = 38) and EG&G Wallac TRFIA (r = 0.926, n = 64). All other quality criteria was also fulfilled with respect to precision, accuracy, and dynamic range.     

Phosphorylation and activation of phospholipase D1 by protein kinase C in vivo: determination of multiple phosphorylation sites.

Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCalpha in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCalpha in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory "loop" region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo.     

Exploring the conformational diversity of loops on conserved frameworks

Loops are structurally variable regions, but the secondary structural elements bracing loops are often conserved. Motifs with similar secondary structures exist in the same and different protein families. In this study, we made an all-PDB-based analysis and produced 495 motif families accessible from the Internet. Every motif family contains some variable loops spanning a common framework (a pair of secondary structures). The diversity of loops and the convergence of frameworks were examined. In addition, we also identified 119 loops with conformational changes in different PDB files. These materials can give some directions for functional loop design and flexible docking.