How cyclic loading affects the migration of radio-opaque markers attached to tendon grafts using a new method: a study using roentgen stereophotogrammetric analysis (RSA)

An increase in anterior laxity following reconstruction of the anterior cruciate ligament (ACL) can result from lengthening of the graft construct in either the regions of fixation and/or the region of the graft substance between the fixations. RSA could be a useful technique to determine lengthening in these regions if a method can be devised for attaching radio-opaque markers to soft tissue grafts so that marker migration from repeated loading of the graft is limited. Therefore, the objectives of this study were 1) to develop a method for attaching radio-opaque markers to an ACL graft that limits marker migration within the graft, 2) to characterize the error of an RSA system used to study migration, and 3) to determine the maximum amount of migration and the time when it occurs during cyclic loading of ACL grafts. Tendon markers were constructed from a 0.8-mm tantalum ball and a stainless steel suture. Ten double-looped tendon grafts were passed through tibial tunnels drilled in bovine tibias and fixed with a tibial fixation device. Two tendon markers were sewn to one tendon bundle of each graft and the grafts were cyclically loaded for 225,000 cycles from 20 N to 170 N. At specified intervals, simultaneous radiographs were obtained of the tendon markers and a radiographic standard of known length. The bias and imprecision in measuring the length of the radiographic standard were 0.0 and 0.046 mm respectively. Marker migration was computed as the change in distance between the two tendon markers along the axis of the tibial tunnel. Marker migration was greatest after 225,000 cycles with a root mean square (RMS) value of less than 0.2 mm. Because the RMS value indicates the error introduced into measurements of lengthening and because this error is small, the method described for attaching markers to an ACL graft has the potential to be useful for determining lengthening of ACL graft constructs in in vivo studies in humans.     

The epidemiology of Leber hereditary optic neuropathy in the North East of England

We performed the first population-based clinical and molecular genetic study of Leber hereditary optic neuropathy (LHON) in a population of 2,173,800 individuals in the North East of England. We identified 16 genealogically unrelated families who harbor one of the three primary mitochondrial DNA (mtDNA) mutations that cause LHON. Two of these families were found to be linked genetically to a common maternal founder. A de novo mtDNA mutation (G3460A) was identified in one family. The minimum point prevalence of visual failure due to LHON within this population was 3.22 per 100,000 (95% CI 2.47-3.97 per 100,000), and the minimum point prevalence for mtDNA LHON mutations was 11.82 per 100,000 (95% CI 10.38-13.27 per 100,000). These results indicate that LHON is not rare but has a population prevalence similar to autosomally inherited neurological disorders. The majority of individuals harbored only mutant mtDNA (homoplasmy), but heteroplasmy was detected in approximately 12% of individuals. Overall, however, approximately 33% of families with LHON had at least one heteroplasmic individual. The high incidence of heteroplasmy in pedigrees with LHON raises the possibility that a closely related maternal relative of an index case may not harbor the mtDNA mutation, highlighting the importance of molecular genetic testing for each maternal family member seeking advice about their risks of visual failure.     

The pedigree rate of sequence divergence in the human mitochondrial genome: there is a difference between phylogenetic and pedigree rates

We have extended our previous analysis of the pedigree rate of control-region divergence in the human mitochondrial genome. One new germline mutation in the mitochondrial DNA (mtDNA) control region was detected among 185 transmission events (generations) from five Leber hereditary optic neuropathy (LHON) pedigrees. Pooling the LHON pedigree analyses yields a control-region divergence rate of 1.0 mutation/bp/10(6) years (Myr). When the results from eight published studies that used a similar approach were pooled with the LHON pedigree studies, totaling >2,600 transmission events, a pedigree divergence rate of 0.95 mutations/bp/Myr for the control region was obtained with a 99.5% confidence interval of 0.53-1.57. Taken together, the cumulative results support the original conclusion that the pedigree divergence rate for the control region is approximately 10-fold higher than that obtained with phylogenetic analyses. There is no evidence that any one factor explains this discrepancy, and the possible roles of mutational hotspots (rate heterogeneity), selection, and random genetic drift and the limitations of phylogenetic approaches to deal with high levels of homoplasy are discussed. In addition, we have extended our pedigree analysis of divergence in the mtDNA coding region. Finally, divergence of complete mtDNA sequences was analyzed in two tissues, white blood cells and skeletal muscle, from each of 17 individuals. In three of these individuals, there were four instances in which an mtDNA mutation was found in one tissue but not in the other. These results are discussed in terms of the occurrence of somatic mtDNA mutations.     

Structure of Escherichia coli 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase inhibitor complexes provide insight into the conformational changes required for substrate binding and catalysis

5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is a key enzyme in a number of critical biological processes in many microbes. This nucleosidase catalyzes the irreversible hydrolysis of the N(9)-C(1') bond of MTA or AdoHcy to form adenine and the corresponding thioribose. The key role of the MTA/AdoHcy nucleosidase in biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing has made it an important antimicrobial drug target. The crystal structures of Escherichia coli MTA/AdoHcy nucleosidase complexed with the transition state analog, formycin A (FMA), and the nonhydrolyzable substrate analog, 5'-methylthiotubercidin (MTT) have been solved to 2.2- and 2.0-A resolution, respectively. These are the first MTA/AdoHcy nucleosidase structures to be solved in the presence of inhibitors. These structures clearly identify the residues involved in substrate binding and catalysis in the active site. Comparisons of the inhibitor complexes to the adenine-bound MTA/AdoHcy nucleosidase (Lee, J. E., Cornell, K. A., Riscoe, M. K., and Howell, P. L. (2001) Structure (Camb.) 9, 941-953) structure provide evidence for a ligand-induced conformational change in the active site and the substrate preference of the enzyme. The enzymatic mechanism has been re-examined.     

Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain

Diacylglycerol (DAG) lipase activity is required for axonal growth during development and for retrograde synaptic signaling at mature synapses. This enzyme synthesizes the endocannabinoid 2-arachidonoyl-glycerol (2-AG), and the CB1 cannabinoid receptor is also required for the above responses. We now report on the cloning and enzymatic characterization of the first specific sn-1 DAG lipases. Two closely related genes have been identified and their expression in cells correlated with 2-AG biosynthesis and release. The expression of both enzymes changes from axonal tracts in the embryo to dendritic fields in the adult, and this correlates with the developmental change in requirement for 2-AG synthesis from the pre- to the postsynaptic compartment. This switch provides a possible explanation for a fundamental change in endocannabinoid function during brain development. Identification of these enzymes may offer new therapeutic opportunities for a wide range of disorders.     

Normal hematologic and serum clinical chemistry values for captive chimpanzees (Pan troglodytes)

In the study reported here, reference intervals for hematologic and serum clinical chemistry variables in the chimpanzee (Pan troglodytes) were developed and characterized. Data were collected longitudinally across a 10-year period for 86 subjects at the Primate Foundation of Arizona (PFA). Variables included nine standard hematologic and 25 standard serum clinical chemistry values. An analysis of variance (ANOVA) was used to test for main effects by age and sex. In addition, PFA mean and range values were compared with those published for humans and six other chimpanzee colonies. The ANOVA results suggest an age effect on hematologic (mean corpuscular hemoglobin, mean corpuscular volume, neutrophils) and serum clinical chemical (creatinine, total protein, globulin, tryglycerides, direct bilirubin, iron, (gamma-glutamyltransferase, alanine transaminase, creatine kinase) values. In addition, sex had a main effect on several variables (red blood cells, hemoglobin concentration, hematocrit, uric acid and sodium concentrations, and aspartate transminase and creatine kinase activities); values for males were greater than those for females. Further, human and chimpanzee mean and range values often were indistinguishable from one another. However, changes in human and chimpanzee values associated with age differ and suggest that hematologic and serum clinical chemistry values may be differentially affected by physical and sexual maturation in humans and chimpanzees.     

Developmental events and shoot apical meristem gene expression patterns during shoot development in Arabidopsis thaliana

Critical developmental and gene expression profiles were charted during the formation of shoots from root explants in Arabidopsis tissue culture. Shoot organogenesis is a two-step process involving pre-incubation on an auxin-rich callus induction medium (CIM) during which time root explants acquire competence to form shoots during subsequent incubation on a cytokinin-rich shoot induction medium (SIM). At a histological level, the organization of shoot apical meristems (SAMs) appears to occur during incubation on SIM about the time of shoot commitment, i.e. the transition from hormone-dependent to hormone-independent shoot development. Genes involved in SAM formation, such as SHOOTMERISTEMLESS (STM) and CLAVATA1 (CLV1), were upregulated at about the time of shoot commitment, while WUSCHEL (WUS) was upregulated somewhat earlier. Genes required for STM expression, such as CUP-SHAPED COTYLEDON 1 and 2 (CUC1 and 2) were upregulated prior to shoot commitment. Gene expression patterns were determined for two GFP enhancer trap lines with tissue-specific expression in the SAM, including one line reporting on CUC1 expression. CUC1 was generally expressed in callus tissue during early incubation on SIM, but later CUC1 was expressed more locally in presumptive sites of shoot formation. In contrast, the expression pattern of the enhancer trap lines during zygotic embryogenesis was more localized to the presumptive SAM even in early stages of embryogenesis.     

Crystal structure and functional analysis of the histone methyltransferase SET7/9

Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.