In vivo measurement of the dynamic 3-D kinematics of the ovine stifle joint

The ovine stifle joint is a promising animal model for investigation of joint mechanobiology. A method for in vivo measurement of dynamic 3-D kinematics of the ovine stifle joint is described (accuracy: 0.36 +/- 0.39 mm). Inter-subject variability in kinematics is greater than both intra-subject and inter-session variability. For future studies in which joint kinematics are measured prior to and following controlled orthopaedic interventions, pooling of data should be avoided and each subject should act as its own control.     

ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization

Many point mutations in human Cu,Zn superoxide dismutase (SOD) cause familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder in heterozygotes. Here we show that these mutations cluster in protein regions influencing architectural integrity. Furthermore, crystal structures of SOD wild-type and FALS mutant H43R proteins uncover resulting local framework defects. Characterizations of beta-barrel (H43R) and dimer interface (A4V) FALS mutants reveal reduced stability and drastically increased aggregation propensity. Moreover, electron and atomic force microscopy indicate that these defects promote the formation of filamentous aggregates. The filaments resemble those seen in neurons of FALS patients and bind both Congo red and thioflavin T, suggesting the presence of amyloid-like, stacked beta-sheet interactions. These results support free-cysteine-independent aggregation of FALS mutant SOD as an integral part of FALS pathology. They furthermore provide a molecular basis for the single FALS disease phenotype resulting from mutations of diverse side-chains throughout the protein: many FALS mutations reduce structural integrity, lowering the energy barrier for fibrous aggregation.     

Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (10(4), 10(6), 10(8), and 10(9) infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (10(9) IU), or saline. Transduction with rAAV-IL-10 at 10(9) IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 10(8) IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.     

The design and synthesis of purine inhibitors of CDK2. III

Cyclin-dependent kinases (CDKs) belong to a class of enzymes that control the ability of a cell to enter into and proceed through the cell division cycle. Using purine as a scaffold, we have synthesized a number of nanomolar inhibitors of CDK-2/cyclin E. In this report, the synthesis of a series of piperidine-substituted purine analogs will be presented, as well as some of their in vitro and in vivo biological effects.     

Estimation of directional division frequencies in vascular cambium and in marginal meristematic cells of plants

Using simple arithmetical formulae, it is shown that, when the meristematic initial cells of a growing plant organ are arranged in a ring, the cellular dimensions predict the relative frequencies of anticlinal and periclinal divisions which these cells undergo. The pattern of cell file branching which appears during the course of development, and which is predicted by this mathematical model, is validated using data pertaining to the numbers and dimensions of initial cells within the secondary vascular cambium of hybrid aspen trees. Data pertaining to a second, simpler set of initial cells which comprises the outer cellular ring of the thallus of the alga Coleochaete orbicularis, and from which all the radial cell files of the circular disc-like thallus are descended, have also been used for model validation. Combining the mathematical approach to division frequencies with data of actual cell sizes permits inferences about the course of the increase of the number of cell files (generated by the anticlinal divisions) and the number of cells within each file (generated by the periclinal divisions) during the earlier stages of secondary tissue or thallus development, and also about how they will develop at future stages. The question whether or not cell division patterns conform to the geometry of the system in which the cells are embedded is also discussed.     

Aged men display blunted biorhythmic variation of muscle performance and physiological responses.

Aging is known to disrupt the "biological clock" that governs physiological variables at rest. This study sought to determine whether aged men demonstrated biorhythmic variation in muscle performance during resistance exercise and physiological responses to that stimulus. Ten aged (75.6 +/- 1.6 yr; mean +/- SE) men completed an isokinetic testing protocol of knee extensors and flexors at 0800, 1200, 1600, and 2000 h. Although time of day variation in peak torque was detectable, significant (P < or = 0.05) oscillation was established only in the knee flexors at 3.14 rad/s. Heart rate, blood pressure, and rectal temperature displayed no significant variation, but trends (P < 0.10) in oscillation of postexercise blood pressure and rectal temperature were noted. Temporal patterns in biorhythmic variation of muscle performance, as well as thermal and cardiovascular measures, emulated those observed in a previous study involving young men where the magnitude of variation was sufficient to achieve statistical significance. Similar to our earlier findings in young men, however, pre- and postexercise testosterone and cortisol concentrations demonstrated significant variation among aged men. These data confirm the blunting of biorhythmic variation in muscle performance and physiological variables, except for circulating hormones, in aged men.     

Applications of NMR to structure-based drug design in structural genomics

Structural genomics is poised to have a tremendous impact on traditional structure-based drug design programs. As a result, there is a growing need to obtain rapid structural information in a reliable form that is amenable to rational drug design. In this manner, NMR has been expanding and evolving its role in aiding the design process. A variety of NMR methodologies that cover a range of inherent resolution are described in the context of structure-based drug design in the era of structural genomics.     

Inhibition of arginine gingipains (RgpB and HRgpA) with benzamidine inhibitors: zinc increases inhibitory potency

We assayed several benzamidine derivatives for inhibition potency with HRgpA and RgpB gingipains, enzymes which are involved in the pathogenesis of gingivitis and periodontal disease. The benzamidine derivatives proved to be effective inhibitors of HRgpA and RgpB, with the best inhibitor being a bis-benzamidine with a urea linker (Ki=30 microM). The inhibition potency was increased 2-3 fold in the presence of low concentrations of zinc with the benzamidines containing a urea moiety linking the two aromatic rings. We propose an inhibition model involving a tetrahedral zinc atom coordinated with the active site Cys and His of gingipain and the urea linker in the benzamidine inhibitor. In summary, we have discovered a new series of effective inhibitors for the gingipains and found a novel way to increase inhibitor potency with the HRgpA and RgpB gingipains using zinc.     

Effect of Rainbow Trout Growth Hormone Complementary DNA on Body Shape,Carcass Yield,and Carcass Composition of F1 and F2 Transgenic Common Carp (Cyprinus carpio

The effect of rainbow trout growth hormone complementary DNA on body shape, dress-out yield, and body composition were assessed in the F1 and F2 generations of transgenic common carp (Cyprinus carpio). All measurements were compared with those for nontransgenic full-sibling common carp in their respective families, and the fish were communally evaluated in earthen ponds. The body weight and length were highly correlated (P <0.01) in both genotypes in all the families. Head morphometrics were negatively correlated (P <0.05) to weight and length of the fish. Various head, body, and caudal traits grew disproportionately faster in transgenic fish in both generations. The altered body shape of transgenic fish resulted in improved dressing percentage in the F2 generation. The carcass composition of transgenic muscle had a lower percentage of (P <0.01) moisture and lipids and higher (P <0.01) percentage of protein in both generations. Six of the 18 amino acids analyzed in F1 transgenic common carp muscle were higher F1 (P <0.05) than the control genotype; however, amino acid ratios were minimally changed. Also, the fatty acid profiles of both genotypes were minimally altered. Higher histidine and lysine ratios in the diet are recommended for maximum growth and health of transgenic common carp in intensive culture systems on the basis of essential amino acid ratios.