Comparison of the linear finite element prediction of deformation and strain of human cancellous bone to 3D digital volume correlation measurements.

The mechanical properties of cancellous bone and the biological response of the tissue to mechanical loading are related to deformation and strain in the trabeculae during function. Due to the small size of trabeculae, their motion is difficult to measure. To avoid the need to measure trabecular motions during loading the finite element method has been used to estimate trabecular level mechanical deformation. This analytical approach has been empirically successful in that the analytical models are solvable and their results correlate with the macroscopically measured stiffness and strength of bones. The present work is a direct comparison of finite element predictions to measurements of the deformation and strain at near trabecular level. Using the method of digital volume correlation, we measured the deformation and calculated the strain at a resolution approaching the trabecular level for cancellous bone specimens loaded in uniaxial compression. Smoothed results from linearly elastic finite element models of the same mechanical tests were correlated to the empirical three-dimensional (3D) deformation in the direction of loading with a coefficient of determination as high as 97% and a slope of the prediction near one. However, real deformations in the directions perpendicular to the loading direction were not as well predicted by the analytical models. Our results show, that the finite element modeling of the internal deformation and strain in cancellous bone can be accurate in one direction but that this does not ensure accuracy for all deformations and strains.

Matrix concentration of insulin-like growth factor I (IGF-I) is negatively associated with biomechanical properties of human tibial cancellous bone within individual subjects.

Insulin-like growth factor-I (IGF-I), abundant in bone matrix, is believed to play an important role during bone development and remodeling. To our knowledge, however, few studies have addressed the relationship between the concentration of IGF-I in bone matrix and the biomechanical properties of bone tissue. In this study, forty-five cylindrical specimens of cancellous bone were harvested from six human tibiae and scanned using micro-computed tomography (microCT). The bone volume fraction (BV/TV) was calculated from three-dimensional (3D) microCT images. Mechanical tests were then performed on a servohydraulic testing system to determine the strength and stiffness of cancellous bone. Following mechanical testing, the concentration of IGF-I in bone matrix was measured by using an enzyme-linked immunoabsorbent assay (ELISA). Within each subject, the concentration of IGF-I in bone matrix had significant (P<0.01) negative correlations with the bone volume fraction, strength, and stiffness of cancellous bone. In particular, the anterior quadrant of the proximal tibia was significantly (P<0.02) greater in IGF-I matrix concentration and marginally significantly lower in strength (P=0.053) and stiffness (P=0.059) than the posterior quadrant. The negative correlations between the cancellous bone matrix concentration of IGF-I and cancellous bone biomechanical properties within subjects found in this study may help us understand the variation of the biomechanical properties of cancellous bone in proximal human tibiae.

N- and C-domains of HIV-1 gp41: mutation, structure and functions.

Recent studies demonstrated that the N- and C-domains of HIV-1 gp41 is involved in virus-mediated membrane fusion resulting in HIV-entry into the target cells. Up to now, viral mutation baffled many scientists to develop effective vaccines and drugs against HIV-1. To acquire more information of mutation of gp41 and to reveal the relationship of structure and function of the N- and C-domains, we compared and analyzed amino acid sequences of the gp41 ectodomain (aa 512-681) of 862 isolates from most HIV-1 clades (including A, B, C, D, E, F, G, H, I, J and O clades). A consensus sequence of the ectodomain with the highest frequency emerging on each position is constituted. The fusion domain and the N-domain belong to the most conserved regions in gp41, and most variable residues assemble partial to the C terminal of gp41. The hydrophobicity of each position is also calculated. The a and d positions in the N-domain for maintaining stabilization of the trimeric coiled coil interactions are highly conservative, and the e and g positions in the C-domain to retain the interaction show also highly conservative. The strange high conservation of the c residues may have an implication in the coiled coil structure. The highly conserved residues form the lining of the hydrophobic cavity and the deep cavity is an ideal target for small molecular inhibitors. On the C-terminal of the C-domain there is a highly conserved segment GIVQQQ. They are intimately involved in forming the three interfaces between neighboring helices. The function of the N- and C-domains, such as binding to the potential cellular receptor and inducing protective activities, are also discussed. These studies on the mutation, structure and functions of the N- and C-domains suggested that both domains become a new focus to develop effective vaccine and antiviral drugs in the new strategies.

ELNKWA-epitope specific antibodies induced by epitope-vaccine recognize ELDKWA- and other two neutralizing-resistant mutated epitopes on HIV-1 gp41.

Based on the fact that monoclonal antibody (mAb) 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 separately or in combination with other mAbs showed potent neutralizing activity to a wide range of primary HIV-1 isolates in vivo and in vitro, but this epitope undergoes restricted mutation. ELNKWA is a neutralizing-resistant mutated epitope. We induced ELNKWA-epitope-specific polyclonal and monoclonal antibodies and studied the interaction of the antibodies with ELDKWA-epitope and other two neutralizing-resistant mutated epitopes. The candidate ELNKWA-epitope-vaccine induced a high level of antibodies to the ELNKWA-epitope-peptide. The ELNKWA-epitope-specific polyclonal antibodies bound not only the ELNKWA-, but also ELDKWA-, ELEKWA- and ELDEWA-epitope-peptides in ELISA-assay. Moreover, the antibodies also recognized four C-domain-peptides (P5, P6, P7, P8) which contain these four epitopes, respectively. Interestingly, an ELNKWA-epitope-specific monoclonal antibody (TH-Ab1) induced by the candidate ELNKWA-epitope-vaccine could also recognize the four C-domain-peptides containing ELNKWA-, ELDKWA-, ELEKWA- and ELDEWK-epitopes. These results indicate that the candidate ELNKWA-epitope-vaccine could induce high levels of antibodies, which recognize the neutralizing epitope ELDKWA and three neutralizing-resistant mutated epitopes, suggesting that the candidate ELNKWA-epitope-vaccine may help to overcome the problem of viral escape from neutralization through mutation at D or K position, and may be developed as an effective vaccine with a broad neutralizing activity against HIV-1.

Induction of monoclonal antibody with predefined ELNKWA epitope specificity by epitope vaccine.

Since the hybridoma technique to produce monoclonal antibodies (MAbs) was discovered, thousands of MAbs with predefined protein specificity have been produced, and a natural or recombinant protein as antigen is necessary for inducing MAbs in the conventional hybridoma technique. To induce epitope-specific MAbs, we suggest an epitope vaccine as a new technique to induce MAbs with predefined epitope specificity. ELDKWA was identified as an important neutralizing epitope on HIV-1 gp41. The MAb 2F5, recognizing ELDKWA epitope, has shown broad neutralizing activity to many HIV strains, including primary isolates, but the mutant in ELNKWA epitope results in escape 2F5-based neutralization. To produce MAbs recognizing this mutated epitope for consideration of passive immunotherapy against the mutant bearing the ELNKWA epitope, MAbs with predefined ELNKWA epitope specificity were induced by synthetic epitope-peptide instead of a natural or recombinant gp41 bearing this epitope. Three MAbs were identified to recognize ELNKWA epitope on the synthetic epitope-peptide, and interestingly could bind the recombinant gp41 with ELDKWA epitope in an ELISA assay and immunoblotting analysis.

Differential induction of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes in Arabidopsis thaliana by wounding and pathogenic attack.

We have isolated cDNAs from two distinct genes encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) in Arabidopsis thaliana. Predicted protein sequences from both genes, DHS1 and DHS2, and a potato DAHP synthase gene are highly related, but none shows significant sequence similarity to conserved microbial DAHP synthase proteins. Despite this structural difference, the DHS1 cDNA complements mutations in a yeast strain lacking DAHP synthase activity. DHS1 RNA levels increase in Arabidopsis leaves subjected either to physical wounding or to infiltration with pathogenic Pseudomonas syringae strains. DHS2 RNA levels are not increased by these treatments, suggesting that the DHS1 and DHS2 proteins fulfill different physiological functions. Other enzymes in the Arabidopsis aromatic pathway are also encoded by duplicated genes, an arrangement that may allow independent regulation of aromatic amino acid biosynthesis by distinct physiological requirements such as protein synthesis and secondary metabolism. The presence of amino-terminal extensions characteristic of chloroplast transit peptides on DHS1 and DHS2 suggests that both proteins may be targeted to the chloroplast.

DNA bending near the replication origin of IncFII plasmid NR1.

The DNA replication origin of plasmid NR1 is located approximately 190 base pairs downstream from the 3' end of the repA1 gene, which encodes the essential initiation protein for replication of the plasmid. Restriction endonuclease fragments that contain the NR1 replication origin and its flanking sequences at circularly permuted positions were obtained by digesting oligomers of ori-containing DNA fragments with sets of enzymes that each cut only once in every ori fragment. Polyacrylamide gel electrophoresis of these permuted restriction fragments showed anomalous mobilities, indicating the presence of a DNA bending locus. Through analysis of the relative mobility plots of these permuted fragments, we found one or two possible DNA bending sites located in the intervening region between the repA1 gene and the replication origin of NR1. It seems possible that DNA bending in this region might help to orient the replication origin alongside the repA1 gene, which could contribute to the cis-acting character of the RepA1 initiation protein.

In-vivo studies on the cis-acting replication initiator protein of IncFII plasmid NR1.

Using segment-directed mutagenesis, a temperature-sensitive mutant of the gene that encodes the cis-acting RepA1 initiation protein of the IncFII plasmid NR1 was isolated. The mutant protein was unable to promote initiation of plasmid replication in vivo at 42 degrees C. Both the wild-type and the mutant repA1 genes were cloned separately into the high-expression vector plasmid pAS1. In these pAS1-repA1 derivatives, the transcription of the repA1 gene was under the control of the lambda PL promoter, which was regulated by the temperature-sensitive lambda cI857 repressor protein. The translation initiation of the repA1 mRNA from these derivatives was mediated by the lambda cII Shine-Dalgarno sequence and initiation codon. The yield of 33,000 Mr RepA1 protein detected on SDS/polyacrylamide gels from Escherichia coli cells containing the pAS1-repA1 derivatives was dependent upon whether the newly synthesized RepA1 was capable of interacting in cis with the downstream NR1 replication origin on the cloned DNA fragment. Mutations in the repA1 gene or deletions of the cis origin region dramatically increased the detectable yield of RepA1 protein. Deletion of the NR1 origin region from the pAS1 derivative containing the wild-type repA1 gene enabled the cis-acting RepA1 protein to complement partially the temperature-sensitive repA1 mutant in trans, to increase the copy number in trans of plasmids that contained the NR1 replicon, and to help NR1 derivatives overcome plasmid incompatibility. The trans effects of RepA1 provided by the pAS1-repA1 derivatives that retained the origin in cis were much less significant. RepA1 provided in trans also stimulated the replication of plasmids carrying cloned copies of the NR1 replication origin region regardless of whether the origin was transcribed from an upstream promoter.

Transcriptional pausing in a region important for plasmid NR1 replication control.

The results of in vitro single-round transcription experiments indicated that RNA polymerase pauses during transcription of the leader region that precedes the repA1 gene of IncFII plasmid NR1. Transcription initiated at either of the two transcription promoter sites of the repA1 gene, which encodes the essential replication initiation protein of NR1, was observed to pause in this region. Pausing was specifically enhanced by addition of NusA protein, an Escherichia coli transcription accessory factor. Northern blot RNA-DNA hybridization analysis of repA1 mRNA synthesized in vivo revealed RNA species that had lengths equivalent to those of the in vitro-paused intermediates. The steady-state rate of in vivo repA1 mRNA transcription downstream from the pause sites (measured by quantitative hybridization of pulse-labeled RNA to DNA probes complementary to different segments of repA1 mRNA) was not appreciably affected, which suggests that the pause sites do not promote premature termination of transcription. The pause sites were located between the target sequence within the leader region of the mRNA that interacts with a 91-base countertranscript and the beginning of the repA1 coding sequence. Because the countertranscript is an inhibitor of translation of repA1 mRNA, transcriptional pausing in this region may be an important feature of the regulation of RepA1 synthesis, which is the mechanism by which plasmid NR1 controls its replication.

Incompatibility and IncFII plasmid replication control.

The DNA coding for replication control and incompatibility of the plasmid NR1 serves as a template in vivo and in vitro for RNA transcription in both directions. In the rightward direction, RNA synthesis begins from 2 different promoters, one of which is regulated and the other constitutive. In vivo, each of these transcripts is more than 1,000 nucleotides long, terminating near the estimated site for the origin of replication. These transcripts serve as messenger RNA for several proteins. One protein (repA1) is required for replication and another (repA2) serves as the repressor for the regulated rightward promoter. RNA synthesis in the leftward direction is constitutive and produces a single transcript of 91 nucleotides which is complementary in sequence to the rightward transcripts. This small transcript is the incompatibility product which regulates the replication of the plasmid. When the intracellular concentration of the small transcript is experimentally varied, the rate of translation of the rightward transcripts and the rate of initiation of replication (plasmid copy number) vary inversely to its concentration. The mode of action of this inhibitor RNA is likely to be formation of an RNA-RNA duplex with the rightward transcripts, thereby inhibiting the translation which would produce the required replication protein. The probability that a rightward transcript will escape interaction with the small RNA molecules and thus allow replication to initiate can be predicted from the laws of mass action based on base-stacking free energies for the likely sequences of initial contact. The estimated intracellular RNA concentrations, based on quantitative hybridization experiments, are agreement with those predicted from the calculated equilibrium constants for duplex formation.