Characterization of a Novel cis-3-Hydroxy-l-Proline Dehydratase and a trans-3-Hydroxy-l-Proline Dehydratase from Bacteria.

Hydroxyprolines, such as trans-4-hydroxy-l-proline (T4LHyp), trans-3-hydroxy-l-proline (T3LHyp), and cis-3-hydroxy-l-proline (C3LHyp), are present in some proteins including collagen, plant cell wall, and several peptide antibiotics. In bacteria, genes involved in the degradation of hydroxyproline are often clustered on the genome (l-Hyp gene cluster). We recently reported that an aconitase X (AcnX)-like hypI gene from an l-Hyp gene cluster functions as a monomeric C3LHyp dehydratase (AcnXType I). However, the physiological role of C3LHyp dehydratase remained unclear. We here demonstrate that Azospirillum brasilense NBRC 102289, an aerobic nitrogen-fixing bacterium, robustly grows using not only T4LHyp and T3LHyp but also C3LHyp as the sole carbon source. The small and large subunits of the hypI gene (hypIS and hypIL, respectively) from A. brasilense NBRC 102289 are located separately from the l-Hyp gene cluster and encode a C3LHyp dehydratase with a novel heterodimeric structure (AcnXType IIa). A strain disrupted in the hypIS gene did not grow on C3LHyp, suggesting its involvement in C3LHyp metabolism. Furthermore, C3LHyp induced transcription of not only the hypI genes but also the hypK gene encoding Δ1-pyrroline-2-carboxylate reductase, which is involved in T3LHyp, d-proline, and d-lysine metabolism. On the other hand, the l-Hyp gene cluster of some other bacteria contained not only the AcnXType IIa gene but also two putative proline racemase-like genes (hypA1 and hypA2). Despite having the same active sites (a pair of Cys/Cys) as hydroxyproline 2-epimerase, which is involved in the metabolism of T4LHyp, the dominant reaction by HypA2 was clearly the dehydration of T3LHyp, a novel type of T3LHyp dehydratase that differed from the known enzyme (Cys/Thr).IMPORTANCE More than 50 years after the discovery of trans-4-hydroxy-l-proline (generally called l-hydroxyproline) degradation in aerobic bacteria, its genetic and molecular information has only recently been elucidated. l-Hydroxyproline metabolic genes are often clustered on bacterial genomes. These loci frequently contain a hypothetical gene(s), whose novel enzyme functions are related to the metabolism of trans-3-hydroxyl-proline and/or cis-3-hydroxyl-proline, a relatively rare l-hydroxyproline in nature. Several l-hydroxyproline metabolic enzymes show no sequential similarities, suggesting their emergence by convergent evolution. Furthermore, transcriptional regulation by trans-4-hydroxy-l-proline, trans-3-hydroxy-l-proline, and/or cis-3-hydroxy-l-proline significantly differs between bacteria. The results of the present study show that several l-hydroxyprolines are available for bacteria as carbon and energy sources and may contribute to the discovery of potential metabolic pathways of another hydroxyproline(s).

Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase.

In the aconitase superfamily, which includes the archetypical aconitase, homoaconitase, and isopropylmalate isomerase, only aconitase X is not functionally annotated. The corresponding gene (LhpI) was often located within the bacterial gene cluster involved in L-hydroxyproline metabolism. Screening of a library of (hydroxy)proline analogues revealed that this protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Δ1-pyrroline-2-carboxylate. Furthermore, electron paramagnetic resonance and site-directed mutagenic analyses suggests the presence of a mononuclear Fe(III) center, which may be coordinated with one glutamate and two cysteine residues. These properties were significantly different from those of other aconitase members, which catalyze the isomerization of α- to ß-hydroxy acids, and have a [4Fe-4S] cluster-binding site composed of three cysteine residues. Bacteria with the LhpI gene could degrade cis-3-hydroxy-L-proline as the sole carbon source, and LhpI transcription was up-regulated not only by cis-3-hydroxy-L-proline, but also by several isomeric 3- and 4-hydroxyprolines.

Skeletal stability after mandibular setback surgery: comparison between the hybrid technique for fixation and the conventional plate fixation using an absorbable plate and screws.

PURPOSE: The purpose of this study was to compare the temporal changes in condylar long axis and skeletal stability after sagittal split ramus osteotomy (SSRO) with the hybrid fixation technique and the conventional plate fixation. PATIENTS AND METHODS: Of 44 Japanese patients diagnosed with mandibular prognathism, 22 underwent SSRO with the conventional plate fixation (1 u-HA/PLLA plate and 4 monocortical screws in each side) and 22 underwent SSRO with a hybrid fixation technique (1 u-HA/PLLA plate and 4 monocortical screws and bicortical screw in each side). The temporal changes in condylar long axis and skeletal stability were assessed by axial, frontal, and lateral cephalograms. After surgery, breakage of the plate and screws was checked by 3-dimensional computed tomography (3DCT). RESULTS: Although there was a significant difference between the groups regarding Me-Ag in T1 (P = 0.0138), there were no significant differences between the groups for the other measurements in lateral, frontal and axial cephalometric analysis in each time interval. In two cases, 4 sides in the conventional plate fixation group, failure of the absorbable plate was found by 3DCT. However, there was no breakage in the hybrid fixation group. CONCLUSION: This study suggested that there were no significant differences in the postoperative temporal changes between the two groups in mandibular setback surgery.

Assessment of bone healing and hypoesthesia in the upper lip after Le Fort I osteotomy with self-setting α-tricalcium phosphate and absorbable plates.

PURPOSE: The purpose of this study was to evaluate hypoesthesia of the upper lip and bone formation using self-setting α-tricalcium phosphate (Biopex(®)) between the segments following Le Fort I osteotomy with bent absorbable plate fixation. SUBJECTS AND METHODS: The subjects were 47 patients (94 sides) who underwent Le Fort I osteotomy with and without mandibular osteotomy. They were divided into a Biopex(®) group (48 sides) and a control group (46 sides). The Biopex(®) was inserted into the anterior part of the gap between the segments in the Biopex(®) group. Trigeminal nerve hypoesthesia at the region of the upper lip was assessed bilaterally by the trigeminal somatosensory-evoked potential (TSEP) method. The area of the Biopex(®) at the anterior part in the maxilla was assessed immediately after surgery and 1 year postoperatively by computed tomography (CT). RESULTS: The mean measurable period and standard deviation were 13.2 ± 18.5 weeks in the control group, 14.5 ± 17.9 weeks in the Biopex(®) group, and there was no significant difference in TSEP. The area of the Biopex(®) after 1 year was significantly smaller than that immediately after surgery (right side: P = 0.0024, left side: P = 0.0001) and bone defects between the segments could not be found in the Biopex(®) group. In the control group, although the areas of bone defect after 1 year were significantly smaller than that immediately after surgery on the right side (P = 0.0133) and left side (P = 0.0469) in the frontal view, complete healing of the bone defects could be seen in 12 of 46 sides after 1 year. CONCLUSION: This study suggested that inserting Biopex(®) in the gap between the maxillary segments was useful for new bone formation and it did not prevent the recovery of upper lip hypoesthesia after Le Fort I osteotomy with absorbable plate fixation.

Effect of self-setting α-tricalcium phosphate between segments for bone healing and hypoaesthesia in lower lip after sagittal split ramus osteotomy.

AIMS: The aim of this study was to evaluate hypoaesthesia of the lower lip and bone formation using self-setting α-tricalcium phosphate (Biopex(®)) between the proximal and distal segments following sagittal split ramus osteotomy (SSRO) with bent absorbable plate fixation. SUBJECTS AND METHODS: The subjects were 40 patients (80 sides) who underwent bilateral SSRO setback surgery. They were divided into a Biopex(®) group (40 sides) and a control group (40 sides). The Biopex(®) was inserted into the anterior part of the gap between the segments in the Biopex(®) group. Trigeminal nerve hypoaesthesia in the region of the lower lip was assessed bilaterally using the trigeminal somatosensory-evoked potential (TSEP) method. Ramus square, ramus length, and ramus width, the square of the Biopex(®) at the horizontal plane under the mandibular foramen were assessed preoperatively, immediately after surgery, and 1year postoperatively by computed tomography (CT). RESULTS: The mean measurable period and standard deviation were 9.3±15.7weeks in the control group, 5.3±8.3weeks in the Biopex(®) group, and there was no significant difference. Ramus square after 1year was significantly larger than that prior to surgery and new bone formation was found between the segments in both groups (P<0.05). In the Biopex(®) group, the square of the Biopex(®) after 1year was significantly smaller than that immediately after surgery (P<0.05). CONCLUSION: This study suggested that inserting Biopex(®) in the gap between the proximal and distal segments was useful for new bone formation and it did not prevent the recovery of lower lip hypoaesthesia after SSRO with bent absorbable plate fixation.

Skeletal stability after mandibular setback surgery: comparisons among unsintered hydroxyapatite/poly-L-lactic acid plate, poly-L-lactic acid plate, and titanium plate.

PURPOSE: The purpose of this study is to compare the time-course changes in condylar long-axis and skeletal stability after sagittal split ramus osteotomy (SSRO) with an unsintered hydroxyapatite (u-HA)/poly-L-lactic acid (PLLA) plate, PLLA plate, or titanium plate. PATIENTS AND METHODS: Of 60 Japanese patients diagnosed with mandibular prognathism, 20 underwent SSRO with a u-HA/PLLA plate system, 20 underwent SSRO with a PLLA plate system, and 20 underwent SSRO with a conventional titanium plate system. The time-course changes in condylar long-axis and skeletal stability were assessed by use of axial, frontal, and lateral cephalograms. RESULTS: Compared with the u-HA/PLLA group, the titanium group showed a significantly greater change in the right condyle angle between initially and 1 month (P = .0105) and intercondylar axes angle between 1 and 3 months (P = .0013). The PLLA group showed a significantly greater change than the titanium group (P = .0043) and u-HA/PLLA group (P = .0002) in terms of ramus inclination between 1 and 3 months; however, there were no significant differences among the 3 groups in the other measurements for each time interval. CONCLUSION: This study suggests that there are no significant differences in postoperative time-course changes among a u-HA/PLLA plate system, PLLA plate system, and conventional titanium plate system.

Assessment of bone healing after Le Fort I osteotomy with 3-dimensional computed tomography.

PURPOSE: The purpose of this study was to examine bone healing after Le Fort I osteotomy in Class III patients. PATIENTS AND METHODS: The study group consisted of 18 Japanese patients with mandibular prognathism with and without asymmetry, maxillary retrognathism or open bite. A total of 36 sides were examined. Le Fort I osteotomy was performed without a pterygoid osteotome, with an ultrasonic curette used to remove interference at the pterygomaxillary region. Titanium plates (Universal Mid-face fixation module, Stryker, Freiburg, German) were used for four patients, absorbable plates (poly-L-lactic acid (PLLA): NEOFIX(®), Gunze, kyoto, Japan) were used for four patients and other absorbable plates (uncalcined and unsintered hydroxyapatite and poly-L-lactic acid (uHA/PLLA): super FIXSORB(®)MX, Takiron Co. Ltd, Osaka, Japan) were used for 10 patients, in the same manner. Postoperative computed tomography (CT) was analyzed for all patients pre-operatively and 1 year postoperative. The anterior and lateral areas between the maxillary segments were measured with 3-dimensional (3D) CT. Bone healing at the pterygomaxillary region was also assessed. RESULTS: There were no significant differences in the area of bone defect healing among the plate types. The areas of bone defect after 1 year were significantly smaller than that immediately after surgery on the right side (p=0.0145) and left side (p=0.0010) in the frontal view and right side in the lateral view (p=0.0118). Bone healing at the pterygomaxillary junction was found in all cases without artificial pterygoid plate fracture. Fourteen of 22 sides with artificial pterygoid plate fracture by an ultrasonic curette showed bone continuity between the pterygoid plate and posterior part of maxilla. CONCLUSION: This study suggested that bony healing could occur in spaces between the segments of maxilla and pterygomaxillary regions as well as the region of the anterior and lateral walls in the maxilla, but it is not always complete within 1 year after Le Fort I osteotomy.

An experimental study of use of absorbable plate in combination with self-setting α-tricalcium phosphate for orthognathic surgery.

OBJECTIVE: The purpose of this study was to histologically and immunohistochemically evaluate bone formation using both self-setting α-tricalcium phosphate (α-TCP; Biopex) and absorbable plate (Super Fixsorb-MX) in rabbit cranium bone. STUDY DESIGN: Twelve adult male Japanese white rabbits (12-16 wk, 2.5-3.0 kg) were used. The surgical defects were made in the nasal bone of a rabbit, and Biopex was implanted in the left side and no material in the right side. Two-hole absorbable plate and 2 screws (Super Fixsorb-MX) were fixed across the defect in each side. The rabbits were killed at 1, 4, 12, and 24 weeks after surgery, and formalin-fixed specimens were embedded in acrylic resin. The specimens were stained with hematoxylin and eosin. For immunohistochemical analysis, the specimens were treated with bone morphogenetic protein 2 (BMP-2) antibodies. Finally, these were evaluated microscopically. RESULTS: New bone formation was observed in the region of absorbable plate and nasal membrane after >4 weeks. The area of new bone with Biopex was significantly larger than that of the control side after 1, 4, and 12 weeks (P < .05). The number of BMP-2-stained cells in the experimental side was significantly larger than in the control side after 4 and 12 weeks (P < .05). CONCLUSION: This study suggests that the use of absorbable plate (Super Fixsorb-MX) in combination with Biopex could be useful and that both of Super Fixsorb-MX and Biopex could provide adequate bone regeneration.

Position of mandibular canal and ramus morphology before and after sagittal split ramus osteotomy.

PURPOSE: The purpose of this study was to evaluate changes in the mandibular canal and ramus morphology before and after a sagittal split ramus osteotomy. PATIENTS AND METHODS: The subjects were 30 patients (60 sides) with mandibular prognathism who had undergone bilateral sagittal split ramus osteotomy setback surgery. The mandibular canal position and ramus morphology were measured at the 3 horizontal planes under the mandibular foramen level (level A), 1 cm lower than level A (level B), and 2 cm lower than level A (level C) preoperatively and 1 year postoperatively by computed tomography. RESULTS: Postoperative ramus width, lateral distance, lateral marrow distance, and canal length were significantly larger than the preoperative values at the foramen, 1 cm lower, and 2 cm lower. The mandibular canal completely contacted the lateral cortex without lateral bone marrow in 6 sides (10%) in levels A and B and 4 sides (6.7%) in level C preoperatively and 6 sides (10%) in level C postoperatively. CONCLUSION: This study suggested that postoperative mandibular canal position was located more posteriorly and the postoperative lateral bone marrow became thicker compared with the preoperative state.

Assessment of ramus, condyle, masseter muscle, and occlusal force before and after sagittal split ramus osteotomy in patients with mandibular prognathism.

PURPOSE: The purpose of this study was to examine the relationship between the morphologies of the masseter muscle and the ramus and occlusal force before and after sagittal split ramus osteotomy (SSRO) in patients with mandibular prognathism. PATIENTS AND METHODS: The study group consisted of 26 patients with mandibular prognathism. All patients underwent bilateral SSRO as well as 3-dimensional computed tomography on which the masseter muscle, ramus, and condyle were measured preoperatively and at 1 year postoperation. Occlusal force and contact area were also recorded with pressure-sensitive sheets. RESULTS: In the cross-sectional area of the masseter muscle, there were no significant differences between the pre- and postoperative status. However, postoperative ramus width and area were significantly larger than preoperative values (P < .0001). Postoperative right condylar area was significantly larger than the preoperative value (P = .0120). Occlusal force and contact area 1 year after surgery were significantly larger than the preoperative values (P = .0016, P = .0190). CONCLUSION: This study suggested that the masseter muscle area did not significantly differ from preoperative status 1 year after SSRO, although occlusal force, contact area, and ramus area and width increased significantly 1 year after SSRO.