Isolation of Enterobacter cowanii from Eucalyptus showing symptoms of bacterial blight and dieback in Uruguay.

AIMS: This study was performed to identify bacterial strains isolated simultaneously with Pantoea species from Eucalyptus trees showing symptoms of bacterial blight and dieback in Uruguay. METHODS AND RESULTS: Several molecular techniques including 16S rRNA and rpoB gene sequencing and DNA-DNA hybridization were used to characterize the gram-negative, facultatively anaerobic, slime-producing bacterial strains isolated along with Pantoea species from Eucalyptus. Hypersensitivity reactions (HR) and pathogenicity tests were performed on tobacco and Eucalyptus seedlings, respectively. The isolates clustered closely with the type strain of Enterobacter cowanii in both phylogenetic trees constructed. The DNA-DNA similarity between the isolates and the type strain of Ent. cowanii ranged from 88% to 92%. A positive HR was observed on the tobacco seedlings, but no disease symptoms were visible on the inoculated Eucalyptus seedlings. CONCLUSIONS: Enterobacter cowanii was isolated from trees with symptoms of bacterial blight although strains of this bacterial species do not appear to be the causal agent of the disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first report of Ent. cowanii isolated from Eucalyptus. Its presence in Eucalyptus tissue suggests that it is an endophyte in trees showing symptoms of blight.

Functionally different isoforms of the human calcitonin receptor result from alternative splicing of the gene transcript.

Two subtypes of the human calcitonin receptor (hCTR) have been described which differ from one another by the presence or absence of a 16-amino acid insert in the first intracellular loop. Both isoforms were stably expressed in baby hamster kidney cells to compare their ligand binding and second messenger coupling. The binding affinity and the on/off rate of binding for salmon CT were identical for the two receptor isoforms. However, the presence of the insert significantly reduced the ability of the receptor to couple to both adenylate cyclase and phospholipase C. Stimulation of a transient calcium response was only observed with the insert-negative receptor. Similarly, the ED50 for the cAMP response is 100-fold higher for the insert-positive form compared with the insert-negative form of the receptor. However, the maximal cAMP response was equivalent for both receptor isoforms. The rate of internalization of the insert-positive form of the receptor is significantly impaired relative to the insert-negative receptor, which suggests that this process may be dependent on the stimulation of a second messenger pathway. Cloning and characterization of the relevant portion of the hCTR gene revealed that these isoforms are generated by alternative splicing. We also discovered a third isoform of the hCTR, which can be generated by alternative splicing at the same position. The presence of a stop codon in this newly described alternative exon would lead to premature termination of the receptor at the C-terminal end of the first transmembrane domain.

Divergent structural requirements exist for calcitonin receptor binding specificity and adenylate cyclase activation.

The basis of the high potency of salmon calcitonin (sCT) in radioligand binding competition and cAMP accumulation studies with cloned calcitonin (CT) receptors from rats, pigs, and humans was examined using two sets of CT analogues, i.e., chimeric sCT/human CT (hCT) analogues and analogues of sCT with differing capacities to form an amphipathic alpha-helix. In competition for 125I-sCT binding the following relative specificities were observed for the chimeric peptides: rat C1a CT receptor, sCT > or = (1-16)hCT/(17-32)sCT (ACT-15) > (1-16)sCT/(17-32)hCT (ACT-27); rat C1b CT receptor, sCT >> ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > ACT-27; porcine CT receptor, sCT > ACT-27 > ACT-15. In contrast, in ligand-induced cAMP accumulation studies the relative efficacies were as follows: rat C1a CT receptor, sCT = ACT-15 > ACT-27; rat C1b CT receptor, sCT = ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > or = to ACT-27; porcine CT receptor, sCT = ACT-15 = ACT-27. The data demonstrate that residues present in the carboxyl-terminal half of sCT are more important for binding competition with the rat C1a, rat C1b, and human CT receptors, whereas residues in the amino-terminal half of sCT are more important for binding competition with the porcine CT receptor. Carboxyl-terminal sCT residues are also important for full potency in adenylate cyclase activation with the rat C1a and rat C1b CT receptors but are less important for activation via the hCT receptor. The disparity in the relative potencies of the peptides in studies of binding competition and cAMP accumulation is suggestive of significant differences in the relative affinities of the peptides for active and inactive conformations of the CT receptor. The use of sCT analogues with varying capacities to form alpha-helices also revealed divergence in the responses of different receptors. This was most apparent for the stimulation of cAMP production by the rat receptor isoforms C1a and C1b. In cells expressing the C1a receptor, the helical analogues sCT and des-Ser2-sCT were equipotent with [Gly8]-des-Leu19-sCT and des-1-amino-[Ala1,7,Gly8]-des-Leu19 sCT, analogues that have reduced or absent helical structure, respectively. In contrast, the nonhelical analogues were 100-1000-fold less potent than sCT and des-Ser2-sCT at the C1b receptor. In general, reduction in the ability of sCT analogues to form helix structures had a greater impact on the potency of the analogues in competition for 125I-sCT binding than in cAMP accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium inflow in cells transfected with cloned rat and porcine calcitonin receptors.

Ca2+ fluxes were examined in HEK 293 cells stably expressing the rat or porcine calcitonin receptors (CTRs). Calcitonin (CT) rapidly increased cytosolic Ca2+ ([Ca2+]i) concentrations in these cells in a manner which was sustained in the presence of extracellular Ca2+ ([Ca2+]e). In cells pretreated with CT, elevation of the [Ca2+]e concentration resulted in a further increase in [Ca2+]i which was concentration-dependent with respect to both the concentration of CT and the increment of [Ca2+]e. Untransfected cells, cells transfected with vector alone, and CTR-transfected cells not treated with CT, were unresponsive to [Ca2+]e. The microsomal Ca(2+)-ATPase inhibitor thapsigargin was able to mimic both the acute [Ca2+]i fluxes and responsiveness to [Ca2+]e mediated by CT in these cells. The CT-induced responsiveness to [Ca2+]e was neither mimicked by, nor affected by, activators of the cAMP or protein kinase C pathways. Treatment of cells with pertussis toxin influenced neither the primary Ca2+ fluxes in response to CT or thapsigargin nor the agonist-induced [Ca2+]e influx. Nifedipine failed to block responses to either CT or thapsigargin. These results lead to the important conclusion that the CTR participates in receptor-activated Ca2+ inflow, in which depletion of intracellular Ca2+ pools leads secondarily to influx of extracellular Ca2+.

Truncation of the porcine calcitonin receptor cytoplasmic tail inhibits internalization and signal transduction but increases receptor affinity.

A series of mutant porcine calcitonin receptors with progressively truncated carboxy termini have been expressed in COS and HEK 293 cells. All forms of the receptor, including those totally lacking the cytoplasmic tail, were able to bind 125I-labeled salmon calcitonin. However, removal of C-terminal domains resulted in multiple functional changes in the receptor. First, compared with the wild type receptor, affinity of binding of salmon calcitonin was increased for truncated receptors, whether determined in intact transfected cells or in cell membranes. Second, internalization of the ligand-receptor complex was greatly attenuated for mutants truncated by 44 or 83 amino acids but not for an intermediate form truncated by 63 amino acids. Third, truncation affected signal transduction, which for the porcine calcitonin receptor occurs by generation of intracellular cAMP and Ca2+. The magnitude of adenylate cyclase responses was much reduced for the same mutants defective in internalization. Under conditions where expression of each receptor form was approximately equal, the magnitude of intracellular Ca2+ responses was decreased by C-terminal truncation. These results draw attention to the functional significance of the cytoplasmic tail of the porcine calcitonin receptor and suggest intramolecular interactions between the carboxy terminus and other receptor domains and/or cellular regulatory elements.

Isoforms of the rat calcitonin receptor: consequences for ligand binding and signal transduction.

Two rat calcitonin (CT) receptor isoforms; C1a and C1b, are identical except for the presence of a 37-amino acid insert in the second extracellular domain of C1b. The functional consequences of this insert were examined after stable expression of these receptors into HEK-293 cells. In binding competition studies, dissociation of [125I]salmon CT ([125I]sCT) from C1b cells was rapid and complete, in contrast to dissociation from C1a cells, which was slow and incomplete, as seen with other CT receptor preparations. In these studies, C1a receptors displayed high affinity for salmon CT (Kd, 0.5 +/- 1.3 nM) and a slightly lower affinity for pig CT. Human CT competed more weakly for binding of [125I]CT. Although the relative affinities of the ligands were maintained for C1b receptors, the affinity for sCT was lower (Kd, 23 +/- 2 nM) and pig CT was approximately 10-fold less potent than sCT. Human and rat CT failed to compete with [125I]sCT even at 1 microM with the C1b receptor. Both receptors influence multiple effector systems, indicating coupling to multiple G-proteins. The CT peptides activated adenylate cyclase with relative efficacies consistent with the binding competition potencies. In addition, both receptor isoforms mediated a rapid increase in the levels of intracellular calcium after a CT challenge. These results show that an extracellular modification in the rat CT receptor results in altered ligand recognition as well as altered binding kinetics, but does not modify their ability to generate multiple second messengers.

Amylin is an agonist of the renal porcine calcitonin receptor.

The cloned renal porcine calcitonin (pCT) receptor cDNA expressed by transient transfection in COS-1 cells or stable transfection in HEK-293 cells was assayed for interaction with CT, amylin, and CT gene-related peptide. Both [125I]salmon CT ([125I]sCT) and [125I]rat amylin displayed specific binding to transfected cells, and in both cases, pCT and rat amylin were equipotent in competing for binding. sCT was most potent in binding competition assays, whereas human CT and rat or human CT gene-related peptide did not compete. Despite the greater apparent affinity of sCT for receptor binding, sCT, pCT, and rat amylin had similar efficacies in stimulating the production of cAMP in the stably transfected cell line (EC50, 0.5-1.6 x 10(-9) M). These results contrasted with those obtained with the rat C1a CT receptor, for which amylin did not compete for [125I]sCT binding and stimulated cAMP production only at high concentrations. These results show that pCT and amylin interact with similar potencies with the pCT receptor and suggest that amylin may act as a natural ligand for this receptor.

Magnet retained overdentures using the Astra dental implant system.

The Astra Implant System (AIS) is a biologically sound and simple way of providing stability for the complete lower denture where anatomical changes have made retention, stability and comfort difficult to achieve. An ongoing clinical study has been undertaken where 20 patients have been provided with magnet retained overdentures using this implant system. This represented a total of 70 implants placed. Seven implants were removed from four patients (three at exposure, one infection). The remaining implants have been followed up for 12 to 25 months and are considered to have achieved successful osseointegration. All the patients in the study are successfully wearing a lower denture retained by magnets. The overall success rate for the implants placed is 90% but if implants less than 9 mm in length are excluded from the results then the success rate is 97.7%.

Effect of autogenous bone grafts on healing following Le Fort I maxillary osteotomies.

The healing of Le Fort I maxillary osteotomies was studied histologically in 12 adult rhesus monkeys to determine the effect of split autogenous rib grafts overlaying the osteotomy cuts on the lateral maxillary walls. The maxilla was advanced 8 mm in each animal, and examined at varying intervals up to 24 weeks after surgery. The results indicated that there were no obvious differences between the monkeys with and without the rib grafts. Both groups healed with good osseous union. It is concluded that autogenous rib grafts did not enhance bony healing.

Tooth pulp changes following Le Fort I maxillary osteotomy in a primate model.

Le Fort I maxillary 'down-fracture' osteotomy with 8 mm advancement was performed in 15 adult rhesus monkeys. Forty-five tooth pulps were examined histologically at intervals from 0 to 24 weeks after surgery. Cell degeneration occurred in 31% of pulps examined, necrosis in 16% of pulps and osteo-dentine was found in 7% of pulps. Almost half of the teeth examined (47%) showed marked cellular changes, more frequently found in posterior teeth. Other features noted were inflammation (13%) and reactive dentine in pulps (24%). Axons degenerated initially but recovered by 24 weeks. It is concluded that Le Fort I maxillary osteotomy caused pulpal disturbances in an animal model and the extent to which this occurs in patients needs to be carefully monitored.

The versatility of hydroxyapatite blocks in maxillofacial surgery.

There is considerable interest at present in the potential role of calcium hydroxyapatite as a biomaterial for bone augmentation in oral and maxillofacial surgery. Most of the published work has been on the particulate form, mainly in alveolar ridge procedures. However, hydroxyapatite is also available as solid or porous blocks. The blocks are more predictable in their use than are the particles, and are adaptable and versatile. This paper reviews the biological behaviour of the material and discusses the role of hydroxyapatite blocks in alveolar ridge augmentation and orthognathic surgery.

Biologic basis for interpositional autogenous bone grafts to the mandible.

Interpositional autogenous bone grafting procedures were performed in the mandibles of 12 beagle dogs to assess cell survival within the graft and the superiorly repositioned alveolus, and to monitor the remodeling process. Histologic and radiologic results indicated that the grafts were well accepted and that new bone was rapidly laid down on their trabeculae. However, the osteocytes within the autografts generally did not survive. There was no evidence of necrosis of the superiorly displaced alveolus, nor any resorption of its surface cortex, and it rapidly united with the autograft and the mandible to produce a stable structure. This study confirms that the lingual pedicle of soft tissue is adequate to maintain the viability of the superiorly repositioned alveolus or segment and to allow rapid remodeling of the autogenous bone graft.

Augmentation of the edentulous mandible using bone and hydroxyapatite: a comparative study in dogs.

This study compared autogenous bone, freeze-dried homografts, and hydroxyapatite in interpositional grafting of the mandible in beagle dogs. Healing was assessed radiologically and histologically. Results showed that the rate of incorporation of the autografts was greater than that of the homografts, and at both 4 and 12 weeks healing was more advanced. There was also rapid healing around the hydroxyapatite grafts, which became firmly bonded to the surrounding bone. The apatite was not resorbed, and new trabeculae of bone had been deposited onto much of its surface. This investigation confirms that hydroxyapatite is well tolerated and may be a suitable substitute for autogenous bone for augmentation of the atrophic mandible.

Hydroxyapatite as a bone substitute in the jaws.

Densely sintered calcium hydroxyapatite has previously been shown to be biocompatible and stable. Its possible role in jaw surgery was investigated using a clinically analogous animal model. The apatite was implanted into the mandible of twelve dogs for 12 weeks, and the healing assessed radiologically and histologically. The new bone which was deposited directly onto the surface of the implants bonded them firmly to the adjacent tissues. There was no fibrous tissue between implant and bone. This material appears to be a suitable substitute for autogenous bone when used as inert 'space filler' in maxillo-facial surgical procedures.