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.

Elastase +/- soybean trypsin inhibitor dissociation of rat oral mucosa: ultrastructural and oxidative metabolic destructive changes in isolated, epithelial and dermal mitochondria after dissociation.

Epithelial and connective tissue compartments of rat oral mucosa were dissociated after incubation with elastase +/- soybean trypsin inhibitor (SBTI). Elastase + SBTI induced greater ultrastructural damage within the dissociated compartments than elastase alone. The basal lamina remained with the epithelial layer after elastase separation and was destroyed after exposure to elastase + SBTI. Isolated epithelial mitochondria were more severely damaged ultrastructurally after elastase + SBTI separation of the compartment than those prepared after exposure to elastase alone. Isolated fibroblast mitochondria were damage to the same extent after dissociation of the compartment with either medium. Oxidative metabolism and mitochondrial recoveries declined significantly after exposure to either dissociating medium. Cytochrome oxidase activity was significantly greater than succinic cytochrome c reductase in the control and experimental groups. Oxidative metabolism was found to be significantly greater in the connective tissue compartment than the epithelial compartment after dissociation of immature rat oral mucosa. Our data suggests that caution be utilized in assessing cellular viability and oxidative metabolism in tissue compartments immediately after their dissociation by proteolytic enzymes.

Oxidative phosphorylation in rat oral mucosal mitochondria.

Oral mucosal mitochondria were isolated and characterized morphologically by electron microscopy. Polarographic measurements were made of respiration and oxidative phosphorylation in the mitochondrial preparations. ADP:O ratios approaching or slightly exceeding the theoretical maxima and stabilized respiratory control ratios were achieved with malate + glutamate, succinate and ascorbate-N,N,N1N1 tetramethyl-p-phenylenediamine (TMPD) as substrates. Inhibition by rotenone, antimycin A, azide, and cyanide established the classical electron transport chain as the major pathway of mitochondrial respiration. Respiration of the oral mucosal mitochondria was stimulated by DNP in the presence of succinate. DNP-stimulated respiration exceeded that observed in the presence of ADP plus Pi and increasing the concentration of DNP progressively inhibited respiration.

Adenylate cyclase localization in unfixed specimens of rat oral mucosa and isolated mitochondria.

The cytochemical localization of adenylate cyclase (E.C.4.6.1.1) was demonstrated in unfixed rat oral mucosal specimens and isolated mitochondrial fractions by use of a modified cytochemical reaction mixture. The adenylate cyclase reaction product was observed in the mitochondrial cristae of intact epithelial and fibroblast cells, and in isolated mitochondria after a 10-min incubation in the reaction mixture. The validity of the cytochemical adenylate cyclase method was supported by biochemical studies.

Trauma affects cyclic AMP and DNA levels at injured and non-injured distal oral mucosal sites.

Experiments in rats were conducted to test the hypothesis that gingival trauma affects cyclic AMP and DNA levels at the gingival wound, and non-injured distal (gingival, palatal) sites. Cyclic AMP and DNA levels rose and fell in a cyclic fashion during the time (0.5-24 h) periods analyzed. Significant increases in cAMP levels occurred at 8 and 20 h and at 8 and 16 h, respectively, at the wound and non-injured palatal site, peripheral to the wound. Similar increases (not significant) in cAMP levels were also noted at the non-injured gingival contralateral site at the same time intervals. DNA distributions were found to be significantly greater 10 and 16 h after injury at the gingival wound, and distal non-injured gingival and palatal sites.

Healing following simulated fiber retention procedures in rats.

Healing responses of a simulated fiber retention procedure in rats has been presented. In the area of attached, but severed supracrestal fibers, a loss of cells was observed in the initial stages of repair. Subsequently, connective tissue cells migrated into this area as did blood vessels. A very distinct interface was seen at about 1 week after surgery between the advancing soft tissues from the flap wound surface and the attached, but severed supracrestal fibers. By 4 weeks after surgery, such distinction was absent and supragingival fiber bundles appeared continuous from their cemental attachment over to the adjacent alveolar crest. However, no evidence of cementogenesis was seen at this site which suggests that these attached fibers were attached prior to surgery. The position of the new junctional epithelium appeared to be limited to the level of the retained supracrestal fiber fragments. However, in areas where cementum, and thereby attached fibers, had been removed during surgery, an altered epithelial adherence was usually seen. This altered adherence was in the form of a long junctional epithelium or a short junctional epithelium on top of parallel oriented collagen fibers. The latter also appeared to adhere to the tooth surfaces.

Mitotic response in basal cells of the crevicular epithelium to gingival surgery.

A total of 18 periodontal patients participated in this study. After initial preparation, a gingivectomy was performed on the lingual surfaces of posterior teeth with pockets ranging from 3 to 5 mm in depth. Control specimens were obtained at time of gingivectomy. Subsequently, the patients were divided into three groups (Group I, II, III). Each group contained three male and three female patients. The gingivectomy site was biopsied after surgery at 7, 14, and 28 days. The mean mitotic indices of the basal cell layer of the crevicular epithelium were obtained. At 0 day, the mean mitotic index was 6.6, increased to 11.4 at 7 days, and rose to a high value of 11.6 at 14 days. It was reduced to a value of 9.7 at 28 days. From our data, it appeared that the rise in mitotic activity which followed gingival injury did not fall to preoperative levels at 30 days postsurgery.

Alveolar bone remodeling following osseous surgery. A clinical study.

Remodeling of the alveolar bone crest was monitored following periodontal surgery including osseous recontouring at 26 sites in 17 patients, aged 23 to 60 years, with advanced periodontitis. Comparison of measurements obtained at initial surgery with those noted at re-entry showed a reduction in crest height 3 months postsurgery at all sites tested: A) At the interradicular sites a mean reduction of 0.38 mm; B) at the radicular sites a mean reduction of 0.84 min; and C) at the furcation sites a mean reduction of 0.79 mm. A reduction in crest height was also noted 6 months postsurgery at all sites tested: A) At the interradicular sites a mean reduction of 0.23 mm; B) at the radicular sites a mean reduction of 0.55 mm; and C) at the furcation sites a mean reduction of 0.88 mm. The mean bone loss different surfaces in both 3 month and 6 month groups was statistically significant when compared with zero loss, using the t test. Within the limits of this clinical study, it can be concluded that crestal resorption, albeit limited in extent, was the usual remodeling response following osseous recontouring.

Histological evaluation of human intraosseous healing responses to the placement of tricalcium phosphate ceramic implants. I. Three to eight months.

Eight intrabony lesions in four patients were removed en bloc 3 to 8 months after periodontal flap debridement. At the time of debridement, the position of the gingival margin and the most apically located calculus were notched to serve as reference points. All lesions received "Synthograft" implants and lesions healed uneventfully. Even though these were severely involved periodontal sites, clinical measurements at time of block removal demonstrated gingival recession (average = 2.9 mm) and a gain in clinical closure (average = 2.6 mm). Histologically, graft particles were present in each specimen. They were walled off by collagen and did not appear to enhance new attachment nor did they induce an inflammatory infiltrate. Thus, they seemed to act as nonirritating fillers. Microscopically, closure of the lesions demonstrated repair with limited evidence of new connective tissue attachment. Histologic expression of the clinical gain in closure was the result of closure by long epithelial adhesion (long junctional epithelium) and possible linkage of dentinal collagen with gingival fibers at areas of dentinal resorption. These variations in closure were often seen within the same clinical site.

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)

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.

Elimination of epithelium from healing postsurgical periodontal wounds by ultra-low temperature. Initial observations.

Prevention of epithelial migration into healing postsurgical periodontal wounds may enhance connective tissue attachment. This study attempted to destroy selectively epithelium on gingival flap surfaces by ultra-low temperature. Sixteen sites in four young adult cats received subcrestal reverse bevel incisions followed by sulcular incisions. Full thickness periodontal flaps were reflected, the remaining supracrestal soft tissue surrounding the teeth was removed, and exposed root surfaces were curetted. Narrow zones measuring about 4 mm along the coronal margins of the flaps were exposed to ultra-low temperature for 5 seconds using a gas expansion cryoprobe cooled to -81 degrees C and placed at the oral gingival aspects of the flaps. Flaps were then repositioned and sutured. Control sites were sham-operated with the cryoprobe kept at room temperature (25 degrees C). Gingival biopsies were taken at 1 hour and at 2, 4, 8 and 12 days following surgery. Microscopically, 1-hour postfreezing and control specimens appeared similar. At 2 and 4 days, large surface areas of corium were denuded of epithelium or were covered by necrotic debris. Mild inflammatory responses were noted within the corium. At 8 days, the epithelium almost completely covered the corium with some initial formation of crevicular epithelium. After 12 days, crevicular epithelium was observed in all tissue sections. No significant morphologic damage to the connective tissue was noted at any time. We, therefore, concluded that low cryodoses can effectively destroy oral gingival epithelium without causing significant morphologic damage to the underlying lamina propria at this level of observation.

The remodeling of human gingival tissues following gingivectomy.

A total of 30 facial gingivectomies were carried out for reduction of suprabony pockets (mean +/- S.D. preoperative pocket depth 2.8 mm +/- 0.3 mm). Remodeling of the gingival margin was monitored by clinical measurements. The following techniques were utilized: A polyvinyl stent was constructed for each surgical quadrant. This stent covered the occlusal margins of the involved teeth and contained the fixed point of reference. Preoperatively, the following measurements were taken: the distance from the fixed point to (a) the height of the free gingival margin, and (b) the base of the clinical pocket. For control purposes similar measurements were taken at the facial surface of an adjacent tooth. A standard gingivectomy to the base of the clinical pocket was then performed and a periodontal dressing was applied for 1 week. Utilizing the stent (fixed point of reference), measurements were taken of the healing margin and control sites inn the same manner as preoperatively; 1,4,8 and 12 weeks after surgery. Our findings indicate that 12 weeks after gingivectomy, the newly formed free gingival margin was located coronally to the line of incision in all cases. The mean pocket depth at 12 weeks after surgery was 0.7 mm +/- 0.2 mm. However, the mean gain in coronal marginal height was 1.2 mm +/- 0.3 mm. Thus, a clinical coronal pocket closure of about 0.5 mm seemed to have taken place at the soft tissue-tooth interface. Measurements at the nonoperated control site showed no significant variations in crevicular depth during the experimental period. We therefore conclude that the excisional reduction of a crevice to 0 mm depth was altered by gingival remodeling during the healing phase. In our experience, this remodeling took place within 3 months after surgery and clinically appeared as a limited coronal pocket closure and gain of marginal height.

Human clinical and histologic repair responses following the use of citric acid in periodontal therapy.

Seven blocks containing one tooth each were obtained from two patients undergoing treatment for periodontitis. Sixteen weeks prior to block removal, these sites were treated by periodontal flap surgery. In six teeth a citric acid solution (pH = 1) was applied for 2 minutes to the planed root surfaces. The seventh tooth received identical periodontal therapy except that saline was applied to the planed root for 2 minutes. At the end of the experimental period, the blocks were removed and prepared for histologic study. In five out of six citric acid treated specimens, no evidence was observed suggesting that citric acid applications either initiated or accelerated cementogenesis or functional connective tissue attachment at root surfaces previously exposed to periodontal pockets. In one specimen, repair cementum was seen crestally to the alveolar margin. This cementum was deposited on the surface of acellular cementum. Unfortunately, however, the specimen does not allow us to date the deposition of the repair cementum. It thus remains questionable whether this cemental deposition is indeed a repair response to the specific treatment performed.

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.

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.

Healing responses of human intraosseous lesions following the use of debridement, grafting and citric acid root treatment. I. Clinical and histologic observations six months postsurgery.

Three block sections of treated human intraosseous lesions within one individual are presented. These lesions were debrided, filled with autogenous grafts and at two sites the root was treated with citric acid. The treated sites were lightly root planed once a month and the blocks were removed 6 months after surgery. In addition, the root accretions within two lesions were notched with a bur prior to root planing in order to positively identify the position of these root accretions in the histologic sections. Clinically, the lesions closed by marginal shrinkage and limited pocket closure. Histologically, regeneration of new cementum, osteogenesis at bone and graft spicule seams and reformation of functionally oriented ligament fibers were present at sites within lesions where accretions had covered the root. Such a healing phenomenon was observed close to the base of the lesions and seemed to be related to this spatial configuration rather than to the notching of the root itself. No specific increased healing responses could be attributed to citric acid root treatment. Thus, "regeneration" of new attachment is possible within human intraosseous lesions even at root sites previously covered by accretions. However, such responses seem to be limited to areas near the base of the intraosseous lesion.

Periodontal healing following open debridement flap procedures. II. Histologic observations.

Four tooth-containing blocks were obtained from patients being treated for infraosseous lesions of significant depth as part of their periodontal therapy. Treatment consisted of open flap debridement and professional cleansing at least every 4 weeks. Teeth in block were removed for histologic study 4 to 6 months after surgery. Histologic evaluation of the repair process showed pocket closure by epithelial and connective tissue adhesions in the form of an elongated junction epithelium, beneath which parallel-oriented fibers adhered to the root for a limited distance. Apical to this adhesion, functionally inserted fibers were present. Since no cementogenesis was seen at these latter areas, it must be assumed that the inserted fibers were present before the surgery and were not significantly affected by the procedure. No significant evidence of crestal osteogenesis was noted. However, comparison of the clinical osseous profile recorded at the time of surgical debridement with the flat crest seen in two of our cases histologically, suggests that significant crestal resorption had taken place postsurgically in some of these lesions.

Healing responses of human intraosseous lesions following the use of debridement, grafting and citric acid root treatment. II. Clinical and histologic observations: one year postsurgery.

This report details histologic healing responses at intrabony sites within two patients about 1 year after surgery. Treatment consisted of open flat debridement of the lesions. At specific sites, augmenting procedures such as autogenous grafts, allografts, synthetic grafts and citric acid root treatment were utilized. In addition, notches were made through calculus prior to root planing at specific root surfaces. These notches were placed at varying distances from the base of the lesion. Patients were followed postsurgically with frequent maintenance visits. Block sections were removed at the end of the experimental period. Clinical reduction in pocket depth was noted at all treated sites. This reduction consisted of limited pocket closure, marginal gingival recession and repocketing. Histologically, all specimens showed evidence of repair. The most mature repair appeared at sites treated with debridement and autogenous grafts. "Regeneration" of lost periodontal attachment was demonstrated by evidence of cementogenesis, osteogenesis and the presence of functionally oriented ligaments. However, the coronal regeneration appeared spatially limited. Allografts showed a similar, but less mature healing response. Synthetic graft material acted essentially as a "filler" within the defect. Citric acid root treatment did not demonstrate clear evidence of augmentation of the repair process. Of particular note in these human specimens was further histologic demonstration that "regeneration" potential apparently can only take place in close proximity to histologically viable periodontal ligament cells which may act as "donor sites" for coronal "regeneration" of lost periodontal attachment. This histologic response was observed regardless of treatment modalities used.

Human clinical and histologic responses to Durapatite implants in intraosseous lesions. Case reports.

The healing response following implantation of a nonresorbable ceramic (durapatite) into human periodontal osseous defects was evaluated clinically and histologically. Four tooth-containing blocks were obtained from four patients who had received durapatite implants in osseous defects, each exceeding 4 mm in depth. Each patient was seen for 5 to 13 postsurgical maintenance visits. Teeth in block section were removed between 8 weeks and 8 months postgraft surgery. Clinical evaluation of the repair process demonstrated that pocket depth decreased in all four cases. Histological evaluation of the repair process showed no indication of new periodontal attachment, osteogenesis or cementogenesis, in the host tissues adjacent to the graft particles. Pocket closure appeared to occur by means of a long junctional epithelium and connective tissue adhesions. There was minimal or no evidence of inflammation in all sections associated with the implant. The graft material therefore acted as a biocompatible foreign body within the gingival tissue.