Oxygen kinetic isotope effects in soluble methane monooxygenase.

Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridged diiron site that activates dioxygen in the catalytic oxidation of hydrocarbon substrates. Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH(3)CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which produced glycolonitrile (HOCH(2)CN) and uncoupled oxidase activity that led to water formation. The oxygen KIE was measured independently for both the oxygenase and oxidase reactions, and values of 1.0152 +/- 0.0007 and 1.0167 +/- 0.0010 were obtained, respectively. The isotope effects and separate dioxygen binding studies do not support irreversible formation of an enzyme-dioxygen Michaelis complex. Additional mechanistic implications are discussed in the context of previous data obtained from single turnover and steady-state kinetic studies.

HIV-1 rev depolymerizes microtubules to form stable bilayered rings.

We describe a novel interaction between HIV-1 Rev and microtubules (MTs) that results in the formation of bilayered rings that are 44-49 nm in external diameter, 3.4-4.2 MD (megadaltons) in mass, and have 28-, 30-, or 32-fold symmetry. Ring formation is not sensitive to taxol, colchicine, or microtubule-associated proteins, but requires Mg(2+) and is inhibited by maytansine. The interaction involves the NH(2)-terminal domain of Rev and the face of tubulin exposed on the exterior of the MTs. The NH(2)-terminal half of Rev has unexpected sequence similarity to the tubulin-binding portion of the catalytic/motor domains of the microtubule-destabilizing Kin I kinesins. We propose a model wherein binding of Rev dimers to MTs at their ends causes segments of two neighboring protofilaments to peel off and close into rings, circumferentially containing 14, 15, or 16 tubulin heterodimers, with Rev bound on the inside. Rev has a strong inhibitory effect on aster formation in Xenopus egg extracts, demonstrating that it can interact with tubulin in the presence of normal levels of cellular constituents. These results suggest that Rev may interact with MTs to induce their destabilization, a proposition consistent with the previously described disruption of MTs after HIV-1 infection.

Clinical response to the use of the HTR polymer implant in human intrabony lesions.

Fifteen patients, men and women aged 18 to 65 years, were treated for periodontitis. Each patient had at least two bilateral intrabony defects; one was designated the experimental site and the other the control site. Both sites were surgically debrided, but one osseous defect received an HTR polymer implant. Patients were followed for 12 months postoperatively. Maximum pocket reduction occurred within 2 months of initial therapy in both experimental and control sites. Initially, pocket reduction was significantly greater in implanted sites than in control sites, but this difference did not occur subsequently. At the end of the 12 months, pocket reduction responses were similar at control and implanted sites.

Human suprabony healing responses following root demineralization and coronal flap anchorage. Histologic responses in 7 sites.

6 suprabony pockets in 2 adult patients were treated with debridement and citric acid root demineralization. Following these procedures, coronal positioning of the marginal flaps was performed at 2 sites. At 4 sites, the marginal flaps were sutured coronally using orthodontic brackets as anchors. At 1 additional site, root debridement only was performed and the margin was placed at about crestal level. Prior to root planing, all roots were notched both at the level of the gingival margin and the most apically positioned calculus. Blocks were harvested 7 and 18 weeks postsurgery and prepared for histologic analysis. Clinical results showed a gain in probing attachment in both coronally positioned sites (average = 1.8 mm) and coronally anchored sites (average = 4.5 mm). The additional debrided-only site showed a gain in probing attachment of 0.1 mm. Histologically, new cementum with functionally inserted fibers was seen within the calculus notch in all coronally anchored sites. This new attachment was associated with crestal osteogenesis. In the 2 coronally positioned and root demineralized sites, new attachment was seen within the calculus notch in 1 out of the 2 specimens evaluated. Within the limits of the evidence presented, coronal anchorage of flaps enhanced probing attachment and new attachment when used in conjunction with root demineralization.

Human intrabony lesion responses to debridement, porous hydroxyapatite implants and teflon barrier membranes. 7 histologic case reports.

7 vertical lesions at 7 teeth in 3 adults with severe periodontitis were treated using open surgical debridement, porous hydroxyapatite grafts and placement of a barrier membrane. Roots were notched at both gingival margins and deepest visible calculus. Flaps were sutured coronally and patients were placed on 0.12% chlorohexidine gluconate twice daily for 2 weeks, post-surgery. Patients returned frequently for plaque control until block removal at 16 to 28 weeks post-surgery. 1 additional block was harvested after 28 weeks. The latter site received root planing only and closed by epithelial adhesion. In the 7 experimental sites, clinical responses were uneventful, and gain in clinical closure varied from 1.7 to 5.0 mm (average = 3.6 mm). Histologically, 2 sites exhibited closure by a long junctional epithelium. The remaining 5 sites showed gingival recession to be apical to the calculus notch or the calculus notch to be epithelialized. However, apical to the notch and within the osseous crater, cellular cementum deposition was marked as was increased bone mass. The increase in bone mass was the result of osteogenesis within the surrounding graft particles which often fused with osseous seams of the crater. A functionally-oriented PDL was seen usually at these sites.

Redox reactive reagents inhibiting and inactivating choline acetyltransferase.

3-Trimethylammoniomethyl catechol and N,N-dimethylepinephrine (catecholine) are redox reactive reagents which possess quaternary ammonium functional groups and the capacity to inhibit or inactivate choline binding macromolecules which mediate cholinergic neuronal function. Earlier studies reported the synthesis of 3-trimethylammoniomethyl catechol and demonstrated its redox-dependent covalent inactivation of the nicotinic acetylcholine receptor (Nickoloff et al., Biochemistry 24, 999-1007 (1985)]. Here we present the synthesis of catecholine and show that both 3-trimethylammoniomethyl catechol and catecholine are weak noncompetitive inhibitors (Ki = 15 +/- 6 and 25 +/- 4 mM, respectively) of choline acetyltransferase (EC 2.3.1.6). Both agents irreversibly inactivate the enzyme.

Histologic healing responses in human vertical lesions following the use of osseous allografts and barrier membranes.

4 vertical lesions at 3 teeth in 2 volunteer adults with severe periodontitis were treated by open surgical debridement, osseous allografts and barrier membranes. Roots were notched at both gingival margins and the deepest visible calculus and flaps were sutured coronally. Patients were placed on 0.12% chlorhexidine gluconate 2x daily post-surgery, and blocks were harvested 5 to 6 weeks after surgery. No abnormal clinical responses were noted during the observation period. In the 4 sites, the average preoperative pocket depth was 9.4 mm; the post-operative pocket depth averaged 4.9 mm, recession averaged 0.9 mm and gain in clinical closure averaged 3.6 mm. Histologically, new cementum measured coronally-apically at the treated sites varied from 0.0 mm to 1.7 mm, with an average of 1.1 mm. Osseous remodelling and crestal osteogenesis were seen in association with cementogenesis. The newly deposited cementum showed insertion of functional oriented fibers. New attachment was present within 2 out of 4 calculus notches in this sample.

Healing of human suprabony lesions treated with guided tissue regeneration and coronally anchored flaps. Case reports.

Teflon membranes were interposed between soft tissue flaps and roots in 4 human suprabony lesions affecting the mandibular incisor teeth in 1 volunteer patient. 4 suprabony lesions affecting the mandibular incisor teeth were used as comparisons in a 2nd patient. All roots were debrided using ultrasonic and manual instruments and the most apical positioned calculus/site on the facial surface was notched. Flaps were sutured as coronally as possible using orthodontic brackets as anchors. Tissue blocks were removed 2 to 3 months after procedures and the facial aspects of all teeth were examined histologically. New attachment in the form of new "cementum with functionally oriented fibers" was seen within the calculus notch in 3 out of 4 membrane-treated sites and immediately apical to the notch at 1 site. The 4 comparison sites showed no evidence of new attachment within or adjacent to the notch. Rather, the gingival margins were located apical to the notch. Our observations suggest that guided tissue regeneration techniques improved coronal attachment responses in human suprabony lesions within the sample described.

Human clinical and histologic responses to the placement of HTR polymer particles in 11 intrabony lesions.

Eleven intrabony periodontal lesions in five volunteer patients received surgical debridement followed by site implantation of a porous particulate polymeric composite (HTR polymer). These patients were observed over time periods varying from 4 weeks to 26 weeks. At the end of the individual observation periods, treated sites were surgically removed in block for histologic analysis. Clinical observations indicated a reduction in pocket depth following treatment which consisted of both gingival recession and gain in clinical closure. No untoward effects were observed clinically in any treated patient during the observation periods. Histologic responses varied from gain in closure by epithelial adhesion to new attachment of varying magnitude. Such varied responses were seen within the same patient and between patients. Graft particles were present at sites from 4 weeks to 26 weeks after implantation and were surrounded by connective tissue capsules. At the periphery of some particles, limited bone formation was present. The alveolar bed was remodeling, at times surrounding specific particles. In our sample, HTR polymer, therefore, appeared to be a well tolerated synthetic graft material when implanted in human intrabony lesions.

Human histologic responses to guided tissue regenerative techniques in intrabony lesions. Case reports on 9 sites.

9 sites of 8 teeth in 5 adults with severe periodontitis were treated by open surgical debridement followed by placement of 1 of 2 Teflon barrier membranes. Teeth were notched at both gingival margins and deepest visible calculus on the root. Barrier membranes were placed apical to alveolar crest and coronal to gingival notch. Flaps were sutured coronally and patients were placed on 1.2% chlorohexodine gluconate twice daily for 2 weeks, post-surgery. Subsequent to suture removal, patients returned for frequent plaque control until block removal. In order to observe early healing responses, 6 sites were harvested 5 to 8 weeks after surgical treatment. 3 additional sites were removed 14, 22 and 30 weeks respectively after surgical treatment. Histologically, new cementum was seen in a linear direction along root surfaces in 6 out of 9 sites (length of cementum = 0.5 to 1.7 mm). 3 sites showed no evidence of new attachment. At sites of cementogenesis, functionally-oriented fibers were inserted. The osseous seams opposite the new attachment often demonstrated osteogenesis. Regenerative responses were seen with both types of teflon membranes and were present as early as 5 weeks after surgery.

Teaching periodontics. Past patterns and future prospects.

The article presents a brief history of the development of periodontal concepts and their data base. Because of the rapid change in periodontal science and knowledge, the author pleads for periodontal education based on "show, deliberate, choose, and do" in our dental curricula.

Histologic and clinical responses to porous hydroxylapatite implants in human periodontal defects. Three to twelve months postimplantation.

Twelve intrabony periodontal lesions in three volunteers received surgical debridement followed by site implantation of porous hydroxylapatite implants. These patients were followed over a total of a 1-year observation period. Blocks of treated sites were surgically removed at 3 months, 6 months and 12 months after implantation. Clinical observation indicated a reduction in pocket depth consisting of both recession and clinical gain of attachment. No ill effects were observed. Histologic examination of the treated sites showed ossification of the implant pores and the implant periphery as early as 3 months after implantation, which became pronounced 12 months after placement. At times, peripheral ossification linked with crestal osseous seams. This ossification occurred in the presence of an adjacent root covering, long junctional epithelium, and thus there was no new attachment. On the other hand, this graft material offers the potential of increasing new bone mass within a human intrabony lesion.

Human intraosseous healing responses to the placement of tricalcium phosphate ceramic implants. II. 13 to 18 months.

Five intrabony lesions in a 40-year-old healthy, white female patient suffering with severe periodontitis were removed en bloc, 13 to 18 months after flap debridement and graft placement. At the time of debridement, the position of the gingival margin and the most apically located calculus were notched in the root to serve as reference points. All lesions received tricalcium phosphate ceramic implants, and the lesions healed uneventfully. The sites were professionally cleaned frequently during the experimental period. At the time of block removal, clinical measurements demonstrated gingival recession (average = 2.6 mm) and a gain in clinical closure (average = 2.3 mm). Histologic evaluation of step serial sections of these blocks showed limited presence of graft particles. The particles were surrounded by dense connective tissue. They did not induce inflammation, nor did they appear to enhance osteogenesis or cementogenesis. Closure of lesions was essentially done by a long junctional epithelium adhesion and limited evidence of new connective tissue--root attachment. In the 18-month specimen, active root resorption was seen immediately apical to the junctional epithelium at a site demonstrating significant inflammation in some sections. Fibrils apparently arising from the resorbing dentinal wall were seen in these sections. Serial sections of this site showed cellular cementum and functional fiber attachment at the resorption sites. These variations in resorption/repair/regeneration within the same surgical site were seen 18 months postsurgery and thus may reflect local tissue responses to inflammatory fluctuations rather than surgical sequellae.

Healing following devitalization of sites within the periodontal ligament by ultralow temperatures.

Healing of specific rat periodontal ligament (PDL) sites following freezing injury was monitored histologically to evaluate repair potentials of this tissue. Buccal alveolar plates supporting the first mandibular molar in 15 adult rats were surgically exposed. The coronal 3 mm of plate covering the buccal surface of the mesiobuccal root and underlying periodontal ligament and cementum were subjected to ultralow temperature using a gas expansion micro/cryoprobe, diameter 2 mm, cooled to -81 degrees C for 5 seconds. Tissues were repositioned and sutured in layers. Animals were sacrificed at 1, 24 and 72 hours and at 2, 5 and 7 weeks following surgery and mandibles removed for histologic analysis. Microscopic evaluation of 24-hour specimens showed loss of cellular vitality in the PDL, adjacent bone and cementum at experimental sites. Cellularity of the gingiva coronal to the crest, and of the PDL apical to the experimental zone, appeared to be within normal limits. Transition between experimental and nonexperimental sites was abrupt. After 72 hours cells from the PDL and gingiva appeared to migrate into and repopulate the noncellular PDL. Few capillaries surrounded by mesenchymal cells and limited polymorphonuclear neutrophil infiltration were observed within the injured PDL site. Marginal gingival inflammation was minimal. At 14 days, collagen lysis, resorption of alveolar bone and root resorption were evident. In the PDL space, nonfunctionally oriented, cellular connective tissue elements were abundant. At 5 and 7 weeks, root resorption was marked. At this time interval, osteogenesis at seams of nonvital bone, repair cementum at sites of root resorption, and at times, ankylosis, were noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Human gingival attachment responses to subgingival crown placement. Marginal remodelling.

13 teeth in block were extracted from 2 patients. Their facial periodontal condition was essentially within normal clinical limits. Temporary crowns covering the bevel were placed below the base of the crevice 1 to 8 weeks prior to extraction. At time of extraction, all blocks were decalcified, the temporary crown dissolved, and the blocks prepared for histologic examinations using buccolingual cut, step serial sections. Histologic data revealed reformation of a new supracrestal attachment unit within 1 week following crown placement. The reformation of the gingival unit consisted of marginal recession with apical and lateral migration of the junctional epithelium to the level of remaining cementum inserted fibers. With gingival recession and migration of junctional epithelium, resorption of crestal portions of the facial plate occurred. However, periodontal fibers anchored into cementum opposite the resorbed bone were not lysed. Rather, the attached fibrillar ends appeared to interdigitate with fibers from the corium of the facial gingiva at this site, thereby forming a more apically located crestal attachment. This response may be one mechanism of reformation of the gingival attachment unit taking place following mechanical and/or surgical injury to this site and is completed often, within 2 weeks after injury.