Finite element analysis of an osseointegrated stepped screw dental implant.

An osseointegrated stepped screw dental implant was evaluated using 2-dimensional finite element analysis (FEA). The implant was modeled in a cross section of the posterior human mandible digitized from a computed tomography (CT) generated patient data set. A 15-mm regular platform (RP) Branemark implant with equivalent length and neck diameter was used as a control. The study was performed under a number of clinically relevant parameters: loading at the top of the transmucosal abutment in vertical, horizontal, and 45 degrees oblique 3 orientations. Elastic moduli of the mandible varied from a normal cortical bone level (13.4 GPa) to a trabecular bone level (1.37 GPa). The study indicated that an oblique load and elastic moduli of the cortical bone are important parameters to the implant design optimization. Compared with the cylindrical screw implant, the maximum von Mises stress of the stepped screw implant model was 17.9% lower in the trabecular bone-implant area. The study also showed that the stepped screw implant is suitable for the cortical bone modulus from 10 to 13.4 GPa, which is not necessarily as strict as the Branemark implant, for which a minimum 13.4 GPa cortical bone modulus is recommended.

Finite element analysis of four thread-form configurations in a stepped screw implant.

The purpose of this study was to determine the optimal thread form configuration for an experimental stepped screw implant. Two-dimensional finite element analysis was applied to model the experimental stepped screw implant in a standard cross-section of the posterior human mandible digitized from a CT-generated patient data set. Four different thread form configurations: v-thread (V), thin-thread (T), and two square-thread forms of 0.24 mm (S1) and 0.36 mm (S2) thread width were compared under oblique load in normal cortical bone condition. The support-type constraint position changed from middle to the base of the bone segment. In middle support-type constraint position only the thin-thread (T) model demonstrated significantly different stress distribution from the other three models, however, in base support-type constraint position T and S1 models demonstrated significantly different stress distribution from the other two models. The results implies that v-thread (V) or large square-thread (S2) are optimal thread form for the experimental stepped screw implant. While, minimal support constraints allow clearer differentiation of the stress picture between the different stepped screw types at the trabecular bone-implant interface.

Application of finite element analysis in implant dentistry: a review of the literature.

Finite element analysis (FEA) has been used extensively to predict the biomechanical performance of various dental implant designs as well as the effect of clinical factors on implant success. By understanding the basic theory, method, application, and limitations of FEA in implant dentistry, the clinician will be better equipped to interpret results of FEA studies and extrapolate these results to clinical situations. This article reviews the current status of FEA applications in implant dentistry and discusses findings from FEA studies in relation to the bone-implant interface, the implant-prosthesis connection, and multiple-implant prostheses.

Properties of a recombinant chimeric protein in which the gamma-carboxyglutamic acid and helical stack domains of human anticoagulant protein C are replaced by those of human coagulation factor VII.

A chimeric cDNA, encoding residues 1-46 (the gamma-carboxyglutamic acid module and its trailing helical stack) of human coagulant factor (f) VII, bound to residues 47-419 of human anticoagulant protein C (PC), was constructed and expressed. The resulting protein, r-[delta GD-HSPC/[symbol: see text] GD-HSfVII]PC, was properly processed with regard to signal/propeptide release, cleavage of the K156R dipeptide, Gla and Hya contents, and the presence of glycosylation. The mutant protein displayed normal dependencies on Ca2+ for adoption of its metal ion-dependent conformation and for binding to acidic phospholipid vesicles. The chimera failed to recognize a monoclonal antibody (MAb) specific for the Ca(2+)-induced conformation of the Gla domain (GD) of PC, but did react with another MAb directed in part to the Ca(2+)-dependent conformation of the GD of fVII. Further, this chimeric protein possessed similar steady state constants as wild-type r-PC toward activation by thrombin and thrombin/thrombomodulin. The activated form of the chimera was very similar to that of its wild-type counterpart in its whole plasma anticoagulant activity, as well as its activity toward inactivation of coagulation factor VIII. The chimeric protein did not bind to the fVII cofactor, tissue factor, showing that the GD/HS domain region of fVII is insufficient for that particular interaction. The results demonstrate that the GD/HS of fVII, when present in the PC and APC background, serves to maintain the Ca2+/PL-related functions of these latter proteins, and suggest that the Ca2+ and PL-dependent interactions of the GD-HS of PC are sufficiently general in nature such that the GD-HS regions of other proteins of this type can satisfy most of the requirements of PC and APC. The data presented also offer support for the independent nature of the domain unit consisting of the GD/HS module.

The functions of the first epidermal growth factor homology region of human protein C as revealed by a charge-to-alanine scanning mutagenesis investigation.

Variant proteins containing charge-to-alanine mutations of single amino acid residues and clusters of such groups contained in the epidermal growth factor 1 (EGF1) homology unit of human protein C (PC) have been accomplished, resulting in the following recombinant (r) mutant proteins: r-[E56A/H57A]PC; r-[H66A]PC; r-[D71A]PC; r-[D79A/R81A]PC; r-[E85A/R87A]PC; and r-[R91A/E92A]PC. Studies of the mutant proteins with a variety of Ca2+-dependent and Ca2+-independent monoclonal antibodies not only led to identification of the epitopes of these antibodies, but also confirmed the importance of D/beta-hydroxyaspartic acid (Hya)71 as one probable coordination site for Ca2+. Employing these antibodies, it was also revealed that Ca2+ binding to its site in the EGF1 region of PC did not influence Ca2+ binding or adoption of the Ca2+-dependent conformation of the gamma-carboxyglutamic acid domain of this same protein. In addition, the Ca2+-induced inhibition of PC activation by thrombin, and the kinetic constants for activation of PC by the thrombin/thrombomodulin complex, were only modestly affected by any of the mutations. The mutants r-[E56A/H57A]APC and r-[H66A]APC displayed at least 70% of wild type r-APC activity in a fVIII inactivation assay, while r-[D79A/R81A]APC, r-[E85A/R87A]APC and r-[R91A/E92A]APC possessed only approximately 40% activity in that same assay. The special role of D/Hya71 in this process was confirmed by showing that r-[D71A]APC was inactive in the fVIII-inactivation assay. These findings demonstrate that some of the charged residues of EGF1, most notably those in the carboxy-terminal region of this domain, participate as partial determinants of the anticoagulant activity of APC. Overall, with the exceptions noted, the data generally suggest that the charged residues of the EGF1 domain of PC, and the Ca2+ binding site contained within this module, are likely more involved with maintenance of the overall structural integrity of this module rather than with its direct functional interactions with effectors, activators, or substrates of PC and APC. Lastly, functional Ca2+ binding to the Gla domain of PC is not significantly influenced by the binding of Ca2+ to the EGF1 module.

Functional consequences of mutations in amino acid residues that stabilize calcium binding to the first epidermal growth factor homology domain of human protein C.

Charge-to-alanine mutations of three amino acid residues, viz, D46, D48, and D/Hya71, which are known to be important in stabilizing Ca2+ binding to epidermal growth factor (EGF) domains of vitamin K-dependent blood coagulation proteins, have been engineered into recombinant human protein C (r-PC). The resulting variants were then employed to assess the importance of this Ca2+ binding site in the activation properties of r-PC and in the activity of activated protein C (APC). Another mutation, of D48 to E, was constructed in order that a more conservative mutation at the Ca2+ binding site could be similarly examined. The mutant proteins were fully processed with regard to proper signal peptide cleavage, gamma-carboxylation, and beta-hydroxylation, except, of course, for the D71A mutant in this latter case. The D48E variant possessed an additional residue of gamma-carboxyglutamic acid (Gla), showing that E48 was gamma-carboxylated. All of the mutants were reactive against a monoclonal antibody (MAb) specific for a Ca(2+)-dependent epitope within the amino-terminus of the Gla domain of r-PC, demonstrating that a proper Ca(2+)-dependent conformation was adopted in this region of the protein. None of the mutants, except for [D48 gamma]r-PC, were reactive against another Ca(2+)-dependent MAb which possessed specificity for Ca2+ binding to the EGF1 region of PC-this being the area of the protein that contained the mutated residues. These data strongly suggest that the alanine mutations present at D46, D48, and D71 diminished Ca2+ binding to the EGF1 domain of r-PC. Steady state kinetic analysis demonstrated that determinants for the Ca(2+)-dependent inhibition of the thrombin (fIIa)-catalyzed activation of r-PC, and for the kinetic recognition of the fIIa/thrombomodulin complex, were not dependent on the integrity of the Ca2+ sites present in EGF1. The lone exception was [D48 gamma]r-PC, which did not undergo inhibition by Ca2+, an effect likely due to the potential for altered coordination of Ca2+ due to the Gla insertion, rather than to a dependency on D48. Plasma-based anticoagulant assays, as well as individual factor Va and factor VIIIa inactivation assays, showed that only [D71A]r-APC possessed a significantly reduced activity compared to wild-type r-APC. These observations suggest that D/Hya71 is likely an important determinant for activity of APC toward its physiological substrates, factor Va and factor VIIIa.

The entire gamma-carboxyglutamic acid- and helical stack-domains of human coagulation factor IX are required for optimal binding to its endothelial cell receptor.

The minimal region of the gamma-carboxyglutamic acid (Gla) domain of human factor (f) IX that interacted with its putative bovine aortic endothelial cell (BAEC) receptor was examined by chemical synthesis of peptides with sequence counterparts in this region of the protein, and assessment of their relative abilities to compete with fIX for receptor binding. We found that IC50 values (total peptide concentrations needed to achieve 50% inhibition of binding of [125I]-fIX to BAEC) were ca. 18 nM for unlabeled fIX and 23 nM for the peptide consisting of the entire Gla domain/helical stack (HS) region (residues 1-47) of fIX. The peptide containing only the Gla domain of fIX (residues 1-38) displayed an IC50 value of > 500 nM for this same competitive binding, whereas peptides containing sequences present in positions 1-14 and 1-24 of the Gla domain of human fIX did not significantly compete with [125I]-fIX for BAEC binding. We conclude that whereas a specific receptor recognition element is present within residues 1-14 of fIX, as has previously been concluded by others and by us, full expression of this epitope requires its presence within the entire Gla domain and HS for proper folding. All determinants for proper folding of fIX that lead to BAEC receptor binding appear to be present within these two domains.

The propeptides of human protein C, factor VII, and factor IX are exchangeable with regard to directing gamma-carboxylation of these proteins.

The specificity of the propeptide sequence in directing vitamin K-dependent post-translational gamma-carboxylation has been assessed by examination of the extent of processing of chimeric constructs of blood coagulation factor VII (fVII), factor IX (fIX) and protein C (PC). One chimera consisted of a protein in which the gamma-carboxyglutamic acid (Gla)/helical stack domain of PC (amino acid residues 1 to 46) was replaced by that of fIX (residues 1 to 47) in an otherwise intact PC. Another consisted of the same construction of a fVII/PC Gla domain-based mutant protein. The final chimera contained the leader/propeptide sequence of PC (residues -42 to -1) replaced by that of fIX (residues -46 to -1). In each case, all Glu-precursor Gla residues in the Gla domains of the proteins were fully processed to Gla. These results demonstrate that the propeptides of fIX and PC are capable of directing gamma-carboxylation of the Gla regions of either protein, that the propeptide of PC can fully function in gamma-carboxylation of the Gla region of fVII, and further suggest that, with regard to gamma-carboxylation, communications between the propeptides and Gla domains in intact proteins are general in nature.

Characterization of a cDNA encoding murine coagulation factor VII.

The cDNA encoding murine coagulation factor VII (mfVII) was isolated and reconstructed from a lambda Zap cDNA library generated from murine liver mRNA. The cDNA contains 1903 nucleotides spanning 15 bases upstream of the 5'-translation initiation codon, an open reading frame of 1338 nucleotides, 550 nucleotides downstream of the first stop codon and a 3' poly(A) tail. The translation product is composed of a 41-amino acid signal/propeptide region followed by a 405-residue mature protein. The latter is highly homologous to that of human, rabbit, bovine, Rhesus monkey, and canine fVII. All protein domains of hfVII are strictly conserved in mfVII.

Transfer of specific endothelial cell-binding properties from the procoagulant protein human factor IX into the anticoagulant protein human protein C.

A series of recombinant (r) chimeric mutants of human coagulation protein C (PC) and activated protein C (APC) containing replacements of homologous PC domains by those of human coagulation factor IX (fIX) were generated, with the intention of determining whether the specific bovine aortic endothelial cell (BAEC) receptor-binding characteristics of fIX could be incorporated into the chimeric r-PC while maintaining the essential properties of PC and APC. Using a competitive BAEC displacement assay with [125I]fIX, we found that a chimeric r-PC (r[delta PC1-46/delta fIX1-47]PC), consisting of the entire gamma-carboxyglutamic domain ([GDIX], residues 1-38) and helical stack ([HSIX], residues 38-47) of fIX as replacements for these same domains of PC, provided an IC50 for fIX-related BAEC binding of 13 nM, as compared to 10 nM for that of unlabeled fIX. This showed that all of the BAEC tight binding determinants for fIX existed within the [GDIX/HSIX]. Additionally, this chimera reacted to the same extent as fIX with the Ca(2+)-dependent, [GDIX]-specific monoclonal antibody H5B7 and lost its reactivity to a similar antibody specific for the [GDPC], JTC1. A synthetic peptide containing residues 1-47 of fIX also competed effectively (IC50 = 16 nM) with intact fIX for BAEC binding. Displacement of [125I]fIX from BAEC did not occur with a chimera containing the [HSIX] alone or with another mutant protein possessing a replacement of the two epidermal growth factor (EGF) homology regions of r-PC (residues 47-137) with those same domains of fIX.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function assessment of the role of the helical stack domain in the properties of human recombinant protein C and activated protein C.

The role of the helical stack (HS) in defining the properties of human recombinant (r) protein C (PC) and activated protein C (APC) was assessed. To do so, several mutations were made in this region of the molecule and their effects on the proteins examined. Substitution of the entire HS of PC (residues 38-46) by that of human coagulation factor (f) IX (residues 39-47), yielding r-[HSIX]PC, did not result in any substantial changes in the gamma-carboxyglutamic acid domain (GD)-related Ca(2+)-dependent properties of PC or APC, suggesting that the conformation of the HS may play a more dominant role in these Ca(2+)-dependent properties than do the specific amino acids that differ between these two HS regions. On the other hand, the catalytic efficiency of activation of r-[HSIX]PC by the thrombin/thrombomodulin complex was reduced to approximately one-third of that of wtr-PC, a result that demonstrates a specific role for the HS of PC in this activation process. Another mutation, [Ser42-->Pro], was generated in the HS region of r-PC, providing r-[S42P]PC, a change that according to the empirical algorithm based on the Chou-Fasman secondary structure rules, would disrupt the alpha-helical conformation of the HS. The anticoagulant activity of the corresponding r-[S42P]APC was found to be approximately 35% of that of wtr-APC. Because of the lack of any notable effects of this mutation on other GD-related Ca(2+)-dependent properties of r-PC and r-APC, the basis of this anticoagulant activity loss may be due to its nonmaximal alignment with substrate on the PL surface. The results of this study indicate that the role of the HS of r-PC and r-APC is to provide a region of the protein that is needed to assure optimal alignment on the PL or cell surface of the active site of the enzyme with that of the cleavage sites of the substrates, perhaps by functioning as a scaffold for separation of the active site of APC from the PL surface.

Molecular study on the chromosome 15 breakpoints in the translocation t(15; 17) in acute promyelocytic leukemia (APL).

Chromosomal translocation t(15; 17) is a specific marker of acute promyelocytic leukemia (APL). In this study, molecular cloning of the t(15; 17) breakpoint was carried out in a Chinese APL patient. It has been shown that the retinoic acid receptor alpha (RARA) gene, normally located on chromosome 17, was fused with a new transcription unit PML, normally localized on chromosome 15. We have subsequently cloned a portion of the PML gene and generated a panel of probes. A PML gene rearrangement was detected in 33 out of 36 APL cases studied. 24 rearrangements were clustered in a 4.4 kb region, designated here as PMLbcr1 whereas 9 rearrangements were concentrated in a 6.5 kb region, defining another breakpoint cluster region (PMLbcr2). These two types of rearrangement constitute the basis for the heterogeneity of the PML-RARA fusion gene and its possible biological significance remains to be explored.

[Molecular study on the distinct isoforms of PML-RAR alpha fusion gene transcripts in acute promyelocytic leukemia].

The chromosomal translocation t(15; 17) in acute promyelocytic leukemia (APL) results in the production of a fusion gene PML-RAR alpha specific for the disease. Using the retrotranscriptase/polymerase chain reaction (RT/PCR), we cloned several distinct isoforms of PML-RAR alpha fusion transcripts and obtained their nucleic acid sequences. The results showed that the RAR alpha derived sequences were the same in fusion genes while the PML sequences differed as a result of the locations of PML gene breakpoints. The biological significance of this finding should be further studied.

Breakpoint clusters of the PML gene in acute promyelocytic leukemia: primary structure of the reciprocal products of the PML-RARA gene in a patient with t(15;17).

DNA studies of the translocation t(15;17) in acute promyelocytic leukemia (APL) have shown that the retinoic acid receptor alpha (RARA) gene on chromosome 17 is juxtaposed to the promyelocytic leukemia (PML) gene on chromosome 15. The PML breakpoints have been mapped to 3 clusters: bcr1, bcr2, and bcr3. We have examined the PML breakpoint distribution in a series of 33 Chinese patients with APL. Twenty-two patients fell within bcr1, 2 within bcr2, and 9 within bcr3. The primary structure of the reciprocal chromosome translocation joints of one patient and that of their normal counterparts have been determined and compared to those of 2 previously reported cases. These studies revealed possible topoisomerase II cleavage sites close to the breakpoints and suggested implications of DNA attachment sites to nuclear matrix. We propose that these features are relevant to the process of illegitimate recombination generating the translocation.

Localization of the chromosome 15 breakpoints and expression of multiple PML-RAR alpha transcripts in acute promyelocytic leukemia: a study of 28 Chinese patients.

Translocation (15;17)(q22;q12-q21) is a chromosome aberration specifically found in acute promyelocytic leukemia (APL), that generates a chimeric gene between the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor alpha (RARA) gene, on chromosome 17. In the course of molecular investigations of a series of 28 Chinese patients with APL, we have simultaneously used Southern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analysis to characterize the PML gene breakpoints on chromosome 15 and identify PML-RARA fusion transcripts. Our results confirmed the existence of the three recently described bcr1, bcr2, and bcr3 breakpoint cluster regions. In addition, structural data provided by PML-RARA transcripts allowed us to more accurately locate the 3' borders of clusters bcr1 and bcr3. Moreover, our data suggest a preferential localization of the breakpoints within bcr1 and bcr3. The primary structure of a 1.4 kb DNA segment flanking the 5' part of the PML gene and that of the bcr3 cluster (2.1 kb) were also established.

Molecular rearrangements of the MYL gene in acute promyelocytic leukemia (APL, M3) define a breakpoint cluster region as well as some molecular variants.

Genomic DNA probes generated from the retinoic acid receptor alpha (RARA) gene located on chromosome 17 and from the MYL gene located on chromosome 15 were used to study the chromosome 15 breakpoints resulting from the t(15; 17) translocation in 26 patients with acute promyelocytic leukemia (APL). In 20 out of 22 patients with a detectable MYL rearrangement, the breakpoints were clustered within a 4.4 kb segment designated MYLbcr. The two remaining patients exhibited a more 5' rearrangement at about 10 kb upstream of the MYLbcr region, implying the lack of at least one MYL gene exon in the resulting MYL-RARA fusion gene. The variation of chromosome breakpoints within the MYL gene may explain size heterogeneity previously observed in some MYL-RARA fusion transcripts expressed in APL cells.