Validation of Bone Conversion in Osteoconductive and Osteoinductive Bone Substitutes

Bone reconstruction can be performed with an autogeneic graft from various donor regions. Osteoconductive and osteoinductive bone substitutes originate from substances of diverse chemical and morphological types and can have a synthetic or a biological derivation. Alongside autogeneic bone transplants and allogenic and xenogeneic bone implants, alloplastic bone replacements of synthetic or semi-synthetic origin are being used for defect reconstruction. In an animal model in rabbits five bone substitutes and one autogeneic graft were surgically incorporated into identical bone defects (10times 10 mm in size) in six anatomically defined regions of the skull. With scintigraphic and histological methods, the metabolic dynamics of the bone is examined as it reacts to the transplantation of autogeneic bone or to implanted bone replacement material. The different autogeneic, xenogeneic and alloplastic bone replacement materials can be differentiated according to the functional quality of the new tissue and the dynamics of the bone conversion thus induced. In the comparison of mineralized, osteoconductive bone subsitutes (TCP, HA, calcium carbonate ceramics) with demineralized, osteoinductive implants (DBM new, DBM old) and autogeneic bone grafts, the bone inducing matrices show the largest quantity of new bone formation, making possible a volume-constant reconstruction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants

Background

Tryptophan synthase consists of two subunits, α and β. Two distinct subgroups of β chain exist. The major group (TrpEb_1) includes the well-studied β chain of Salmonella typhimurium. The minor group of β chain (TrpEb_2) is most frequently found in the Archaea. Most of the amino-acid residues important for catalysis are highly conserved between both TrpE subfamilies.

Results

Conserved amino-acid residues of TrpEb_1 that make allosteric contact with the TrpEa subunit (the α chain) are absent in TrpEb_2. Representatives of Archaea, Bacteria and higher plants all exist that possess both TrpEb_1 and TrpEb_2. In those prokaryotes where two trpEb genes coexist, one is usually trpEb_1 and is adjacent to trpEa, whereas the second is trpEb_2 and is usually unlinked with other tryptophan-pathway genes.

Conclusions

TrpEb_1 is nearly always partnered with TrpEa in the tryptophan synthase reaction. However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the functional β chain, as TrpEb_1 is absent. The six lineages show a distinctive divergence within the overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in TrpEa that are otherwise conserved for interfacing with TrpEb_1. We suggest that the standalone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established catalytic capability of tryptophan synthase β chains. A coincident finding of interest is that the Archaea seem to use the citramalate pathway, rather than threonine deaminase (IlvA), to initiate the pathway of isoleucine biosynthesis.     

Dissection of inhibitory Smad proteins: both N- and C-terminal domains are necessary for full activities of Xenopus Smad6 and Smad7

Smad6 and Smad7 comprise a subclass of vertebrate Smads that antagonize, rather than transduce, TGF-β family signaling. These Anti-Smads can block BMP signaling, as evidenced by their ability to induce a secondary dorsal axis when misexpressed ventrally in Xenopus embryos. Smad7 inhibits additional TGF-β related pathways, and causes spina bifida when misexpressed dorsally. We have performed structure-function analyses to identify domains of Anti-Smads that are responsible for their shared and unique activities. We find that the C-terminal domain of Smad7 displays strong axis inducing activity but cannot induce spina bifida. The isolated N-terminal domain of Smad7 is inactive but restores the ability of the C-terminus to cause spina bifida when the two are co-expressed. By contrast, the N- and C-terminal domains of Smad6 have weak axis inducing activity when expressed individually, but show full activity when co-expressed. Chimeric analysis demonstrates that the C-terminal domain of Smad7, but not Smad6, can induce spina bifida when fused to the N-terminal domain of either Smad6 or Smad7. Thus, although the C-terminal domain is the primary determinant of the intrinsic activity of Xenopus Anti-Smads, the N-terminal domain is essential for full activity, is interchangeable between Smad6 and 7, and can function in trans.     

Mechanics of the human femoral adventitia including the high-pressure response

Adventitial mechanics were studied on the basis of adventitial tube tests and associated stress analyses utilizing a thin-walled model. Inflation tests of 11 nonstenotic human femoral arteries (79.3 +/- 8.2 yr, means +/- SD) were performed during autopsy. Adventitial tubes were separated anatomically and underwent cyclic, quasistatic extension-inflation tests using physiological pressures and high pressures up to 100 kPa. Associated circumferential and axial stretches were typically <20%, indicating "adventitiosclerosis." Adventitias behaved nearly elastically for both loading domains, demonstrating high tensile strengths (>1 MPa). The anisotropic and strongly nonlinear mechanical responses were represented appropriately by two-dimensional Fung-type stored-energy functions. At physiological pressure (13.3 kPa), adventitias carry ~25% of the pressure load in situ, whereas their circumferential and axial stresses were similar to the total wall stresses (~50 kPa in both directions), supporting a "uniform stress hypothesis." At higher pressures, they became the mechanically predominant layer, carrying >50% of the pressure load. These significant load-carrying capabilities depended strongly on circumferential and axial in-vessel prestretches (mean values: 0.95 and 1.08). On the basis of these results, the mechanical role of the adventitia at physiological and hypertensive states and during balloon angioplasty was characterized.     

Decreased 5alpha-dihydrotestosterone catabolism suppresses T lymphocyte functions in males after trauma-hemorrhage

Trauma-hemorrhage producesprofound immunosuppression in males but not in proestrus females.Prior castration or flutamide treatment of males followingtrauma-hemorrhage prevents immunosuppression, implicating5-dihydrotestosterone for the immunosuppressive effects. 5-Dihydrotestosterone, a high-affinity androgen receptor-binding steroid, is synthesized in tissues as needed and seldom accumulates. The presence of steroidogenic enzymes in T lymphocytes suggests bothsynthesis and catabolism of 5-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5-dihydrotestosterone activity inT lymphocytes of males following trauma-hemorrhage is due to decreasedcatabolism. Accordingly, catabolism of 5-dihydrotestosterone wasassessed in splenic T lymphocytes by examining the activity andexpression of enzymes involved. Analysis showed increased synthesis anddecreased catabolism of 5-dihydrotestosterone in intact male Tlymphocytes following trauma-hemorrhage. In contrast, reduced5-reductase activity and increased expression of17-hydroxysteroid dehydrogenase oxidative isomers suggestinactivation of 5-dihydrotestosterone in precastrated males. Thusour study suggests increased synthesis and decreased catabolism of5-dihydrotestosterone as a reason for loss of T lymphocyte functionsin intact males following trauma-hemorrhage, as evidenced by decreasedrelease of interleukin-2 and -6.

     

Determinants of cardiac Na(+)/Ca(2+) exchanger temperature dependence: NH(2)-terminal transmembrane segments

The cardiacNa⁺/Ca²⁺ exchanger (NCX) in troutexhibits profoundly lower temperature sensitivity in comparison to themammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout andcanine NCX cDNA and expressed in Xenopus oocytes.NCX-mediated currents were measured at 7, 14, and 30°C using thegiant excised-patch technique. By using this approach, the differentialtemperature dependence of NCX was found to reside within theNH₂-terminal region of the molecule. Specifically, we foundthat ~75% of the Na⁺/Ca²⁺ exchangedifferential energy of activation is attributable to sequencedifferences in the region that include the first four transmembranesegments, and the remainder is attributable to transmembrane segmentfive and the exchanger inhibitory peptide site.

     

The human gamma-aminobutyric acid A receptor delta (GABRD) gene: molecular characterisation and tissue-specific expression

Terminal deletions of 1p36 result in a specific and common syndrome characterised by the following: growth delay, distinctive facial anomalies, hearing and visual deficits, heart defects, body asymmetry, moderate to severe psychomotor retardation, epilepsy, and self-abusive behaviour. The human gamma-aminobutyric acid A receptor delta-subunit gene (GABRD) encodes for one of at least 15 ligand-gated chloride channels for gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. Recently we have mapped this gene by radiation hybrid mapping to the critical region of gene loss of the 1p36 deletion syndrome within 1p36.33. The complete complementary DNA (cDNA) sequence of GABRD was generated using assembled sequence of cDNA fragments already available, and 5'-rapid amplification of cDNA ends products. Fine physical mapping of the GABRD gene within this genomic interval was performed by screening bacterial artificial chromosome contigs spanning the critical region of the 1p36 deletion syndrome. The GABRD gene maps immediately proximal to the PRKCZ gene that is located between marker D1S243 and cosmid D1Z2--a region thought to be critical for cognition and speech development. The GABRD gene is expressed most abundantly in brain and has three alternative exons (1A-C) with alternative start codons at the 5'-end. Genomic localisation, function, and expression would suggest that the GABRD gene represents a good candidate for the neurodevelopmental and neuropsychiatric anomalies seen in the 1p36 deletion syndrome.     

The mitochondrial genome of the pufferfish,Fugu rubripes,and ordinal teleostean relationships

The small nuclear genome of the pufferfish, Fugu rubripes (order Tetraodontiformes), makes this species highly interesting for genome research. In order to establish the phylogenetic position of the Tetraodontiformes relative to other teleostean orders that might also have a reduced nuclear genome size, we have sequenced the mitochondrial (mt) genome of the pufferfish. The gene order, nucleotide composition and evolutionary rate of the mt genome of the fugu correspond to those of other teleosts. This suggests that the evolution of this genome has not been affected by the processes that led to the dramatic reduction of the size of the nuclear genome of the fugu. The phylogenetic analyses, which were based on the concatenated amino acid sequences of twelve protein-coding mt genes, placed the fugu among the percomorphs. The affinities between the Tetraodontiformes and either the Perciformes or the Zeiformes were limited, however. The common notion of a separate euteleostean clade remained unsupported. The analyses did not support the traditional systematic understanding that the Clupeiformes constitute a basal teleostean lineage. In addition the findings strongly suggest that three teleostean orders, the Perciformes, Zeiformes and Scorpaeniformes, are paraphyletic.     

Cloning and modeling of CD8 beta in the amphibian ambystoma Mexicanum. Evolutionary conserved structures for interactions with major histocompatibility complex (MHC) class I molecules

Bovine anaplasmosis is a rickettsial disease of world-wide economic importance caused by Anaplasma marginale. Several major surface proteins with conserved gene sequences have been examined as potential candidates for vaccines and/or diagnostic assays. Major surface protein 1 (MSP1) is composed of polypeptides MSP1a and MSP1b. MSP1a is expressed from the single copy gene msp1 alpha and MSP1b is expressed by members of the msp1 beta multigene family. In order to determine if the msp1 genes are conserved, primers specific for msp1 alpha, msp1 beta(1), and msp1 beta(2) genes were synthesized and used to amplify msp1 sequences of A. marginale from tick cell cultures, from cattle during acute and chronic infections and from salivary glands of Dermacentor variabilis. Protein sequences of MSP1a, MSP1b(1) and MSP1b(2) were conserved during the life cycle of the parasite. No amino acid changes were observed in MSP1a. However, small variations were observed in the MSP1b(1) and MSP1b(2) protein sequences, which could be attributed to recombination, selection for sub-populations of A. marginale in the vertebrate host and/or PCR errors. Several isolate-specific sequences were also observed. Based on the information obtained in this study, the MSP1 protein appears to be fairly well conserved and a potential vaccine candidate.     

Differential expression and genetic variation of hepatic messenger RNAs from genetically lean and fat chickens

Although excessive adiposity has become a major drawback in meat type chicken production, few of the genes involved in this process have been characterized so far. In order to identify putative genes involved in adiposity, we performed differential display analysis of RNAs extracted from the liver of divergently selected lean and fat chickens. Twenty-six differential products were selected and purified by single strand conformation polymorphism gel electrophoresis before sequencing and Northern blot analyses. An orthologous sequence of a mammalian cytochrome P450 2C subfamily member was proven to be differentially expressed in the liver of lean and fat chickens and could play an important role in the regulation of adiposity. In mammals, these genes are involved in detoxification of xenobiotics and metabolism of some important biological compounds. Four other genes were found differentially expressed to a lower extent. Some unidentified products were shown to be lean or fat specific, with sequence polymorphism and liver specific expression, strongly suggesting that the related gene could be directly involved in adiposity. Our data indicate that differential display can evidence genes with differential expression and with sequence polymorphism, making this strategy more accurate for differential analysis of messenger RNAs.