In vivo determination of the Achilles tendon moment arm in three-dimensions

Two-dimensional methods have been applied to determine the Achilles tendon moment arm in previous studies, although the talocrural joint rotates in three-dimension. The purpose of this study was to develop a method for determining the Achilles tendon moment arm in three-dimensions (3DMA). A series of sagittal ankle images were obtained at ankle positions of -20°, -10° (dorsiflexed position), 0° (neutral position), +10°, +20°, and +30° (plantarflexed position). The talocrural joint axis was determined as the finite helical axis of the ankle joint over 20° of displacement, and the 3DMA was determined as the shortest distance from the talocrural joint axis to the line of action of the Achilles tendon force. The corresponding 2DMA was determined with the center of rotation method using the images captured on the sagittal plane passing through the mid-point of the medio-lateral width of the tibia. The 3DMA ranged from 35 to 41 mm across various ankle positions and was, on average, 11 mm smaller than 2DMA. The difference between the two measures was attributable primarily to the deviations of the talocrural joint axis from the anatomical medio-lateral direction. The deviations on the coronal plane (21.4±20.7°) and on the transverse planes (14.8±22.6°) accounted for the errors of 1.3 mm and 3.0 mm, respectively. In addition, selecting either a medially or laterally misaligned sagittal-plane image for determining the 2DMA gave rise to error by 3.5 mm. The remaining difference was accounted for by the random measurement error.     

Myelin Basic Protein-Specific TCR/HLA-DRB5*01:01 Transgenic Mice Support the Etiologic Role of DRB5*01:01 in Multiple Sclerosis

Genetic susceptibility to multiple sclerosis (MS) has been linked to the HLA-DR15 haplotype consisting of DRB1*15:01(DR2b) and DRB5*01:01(DR2a) alleles. Given almost complete linkage disequilibrium of the two alleles, recent studies suggested differential roles in susceptibility (DR2b) or protection from MS (DR2a). Our objective was to assess the potential contribution of DR2a to disease etiology in MS using a humanized model of autoimmunity. To assess the potential contribution of DR2a to disease etiology, we created DR2a humanized transgenic (Tg) mice and subsequently crossed them to Tg mice expressing TL3A6, an MS patient-derived myelin basic protein 83-99-specific TCR. In TL3A6/DR2a Tg mice, CD4 Tg T cells escape thymic and peripheral deletion and initiate spontaneous experimental autoimmune encephalomyelitis (EAE) at low rates, depending on the level of DR2a expression. The ability to induce active EAE was also increased in animals expressing higher levels of DR2a. Inflammatory infiltrates and neuronal damage were present throughout the spinal cord, consistent with a classical ascending EAE phenotype with minor involvement of the cerebellum, brainstem, and peripheral nerve roots in spontaneous, as well as actively induced, disease. These studies emphasize the pathologic contribution of the DR2a allele to the development of autoimmunity when expressed as the sole MHC class II molecule, as well as strongly argue for DR2a as a contributor to the CNS autoimmunity in MS.     

Glycosylation of Simian Immunodeficiency Virus Influences Immune-Tissue Targeting during Primary Infection, Leading to Immunodeficiency or Viral Control

Glycans of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) play pivotal roles in modulating virus-target cell interactions. We have previously reported that, whereas SIVmac239 is pathogenic, its deglycosylated essentially nonpathogenic mutant (Δ5G) serves as a live-attenuated vaccine, although both replicate similarly during primary infection. These findings prompted us to determine whether such a polarized clinical outcome was due to differences in the immune tissues targeted by these viruses, where functionally and phenotypically different memory CD4(+) T cells reside. The results showed that Δ5G replicates in secondary lymphoid tissue (SLT) at 1- to 2-log-lower levels than SIVmac239, whereas SIVmac239-infected but not Δ5G-infected animals deplete CXCR3(+) CCR5(+) transitional memory (TrM) CD4(+) T cells. An early robust Δ5G replication was localized to small intestinal tissue, especially the lamina propria (effector site) rather than isolated lymphoid follicles (inductive site) and was associated with the induction and depletion of CCR6(+) CXCR3(-) CCR5(+) effector memory CD4(+) T cells. These results suggest that differential glycosylation of Env dictates the type of tissue-resident CD4(+) T cells that are targeted, which leads to pathogenic infection of TrM-Th1 cells in SLT and nonpathogenic infection of Th17 cells in the small intestine, respectively.     

SnoN Suppresses Maturation of Chondrocytes by Mediating Signal Cross-talk between Transforming Growth Factor-β and Bone Morphogenetic Protein Pathways

Hypertrophic maturation of chondrocytes is a crucial step in endochondral ossification, whereas abnormally accelerated differentiation of hypertrophic chondrocytes in articular cartilage is linked to pathogenesis of osteoarthritis. This cellular process is promoted or inhibited by bone morphogenetic protein (BMP) or transforming growth factor-β (TGF-β) signaling, respectively, suggesting that these signaling pathways cross-talk during chondrocyte maturation. Here, we demonstrated that expression of Tgfb1 was increased, followed by phosphorylation of Smad2, during BMP-2-induced hypertrophic maturation of ATDC5 chondrocytes. Application of a TGF-β type I receptor inhibitor compound, SB431542, increased the expression of Id1, without affecting the phosphorylation status of Smad1/5/8, indicating that the activated endogenous TGF-β pathway inhibited BMP signaling downstream of the Smad activation step. We searched for TGF-β-inducible effectors that are able to inhibit BMP signaling in ATDC5 cells and identified SnoN. Overexpression of SnoN suppressed the activity of a BMP-responsive luciferase reporter in COS-7 cells as well as expression of Id1 in ATDC5 cells and, subsequently, the expression of Col10a1, a hallmark of hypertrophic chondrocyte maturation. siRNA-mediated loss of SnoN showed opposite effects in BMP-treated ATDC5 cells. In adult mice, we found the highest level of SnoN expression in articular cartilage. Importantly, SnoN was expressed, in combination with phosphorylated Smad2/3, in prehypertrophic chondrocytes in the growth plate of mouse embryo bones and in chondrocytes around the ectopically existing hypertrophic chondrocytes of human osteoarthritis cartilage. Our results indicate that SnoN mediates a negative feedback mechanism evoked by TGF-β to inhibit BMP signaling and, subsequently, hypertrophic maturation of chondrocytes.     

28S rDNA haplotypes of males are distinct from those of androgenetic hermaphrodites in the clam Corbicula leana

The clam Corbicula leana exists in two forms, hermaphrodites and males. Our previous study on mitochondrial DNA suggested that the male nuclear DNA might have derived from hermaphrodite C. leana relatively recently. To clarify the origin of males in the clam, sequences of the nuclear 28S rDNA divergent domain (which is 441-444 bp long) in androgenetic hermaphrodites and males and dioecious (bisexual) species were analyzed. Unexpectedly, the nuclear 28S rDNA haplotypes of males and hermaphrodites were distinct. Haplotype network analysis indicated that males and hermaphrodites are reproductively isolated from each other without sharing the same nuclear haplotype. These results support a hypothesis that the egg nuclear genome of androgenetic hermaphrodites is replaced by the male sperm genome, and only males develop after fertilization by a male spermatozoon.     

A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImN^N). Since the ImN^N nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImN^N unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy-β-d-ribofuranosyl)imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImN^N were obtained in good yield.     

Comparison of plasma membrane proteomic changes of Arabidopsis suspension-cultured cells (T87 Line) after cold and ABA treatment in association with freezing tolerance development

The plasma membrane (PM) is the primary site of freezing injury in plants. To determine global changes in PM protein profiles in association with freezing tolerance development, proteome analysis of the purified PM of Arabidopsis suspension-cultured cells (T87 line) was conducted with label-free protein quantification technology. Freezing tolerance of Arabidopsis cells at the lag growth phase (8 d old) increased after cold acclimation (CA) or ABA treatment. Proteome analysis assigned 658 proteins in the PM in total, of which 45.3% (298 proteins) were predicted to have transmembrane domains. They were classified into several functional categories, with the primary categories being proteins in transporters, signal transduction, protein destination and storage, and cell structure. After CA, 271 proteins increased and 111 proteins decreased. ABA treatment resulted in 185 increased and 56 decreased proteins. Of these, 139 increased and 49 decreased proteins were identified in common after both CA and ABA treatment. In addition, there were proteins specifically expressed in cold- (132 increased and 62 decreased) or ABA- (46 increased and 7 decreased) treated cells. Collectively, our results clearly show that (i) responses of the PM proteome to CA and ABA treatment overlap substantially but, at the same time, some proteins exhibited different response patterns in each treatment; and (ii) the majority of ABA-responsive proteins are CA-responsive proteins but not vice versa, suggesting complex interactions of CA and ABA signaling pathways in the PM proteome responses.     

Real-time weather analysis reveals the adaptability of direct sea-crossing by raptors

Many animals seasonally travel between their breeding and wintering grounds. With their advanced mobility, birds often migrate over thousands of kilometres. Recently, satellite-tracking studies have revealed peculiar migration routes for some avian species at a global scale. However, the adaptability of such migration routes has not been clearly demonstrated. Using satellite-tracking data for 33 individuals, we show that the Japanese population of Oriental honey-buzzards (Pernis ptilorhynchus) directly crosses the 650-km-wide East China Sea during their autumn migration, although they fly a longer route around the sea rather than directly crossing it during their spring migration. By applying aerodynamic theory, we show that the buzzards could cross the sea by soaring and gliding flight. Moreover, using a high-resolution meteorological-prediction analysis, we demonstrate that the migratory trajectory of the birds strongly depends on the wind direction at their estimated locations. In the area, northeastern tailwinds blow stably only during autumn. Thermals were abundant ca. 500–1,000 m over the East China Sea in autumn, but that was not the case in spring. We suggest that the autumn-migration route across the East China Sea is likely to have evolved in response to the specific weather conditions over the sea. Animations showing movements of Oriental honey-buzzards and temporal change in weather conditions are available at: , , , , , and .     

Amino acid sequence of Egyptian goose egg-white lysozyme and effects of amino acid substitution on the enzymatic activity

The amino acid sequence of Egyptian goose lysozyme (EGL) from egg-white and its enzymatic properties were analyzed. The established sequence had the highest similarity to wood duck lysozyme (WDL) with five amino acid substitutions, and had eighteen substitutions difference from hen egg-white lysozyme (HEL). Tyr34 and Gly37 were found at subsites E and F of the active site when compared with HEL. The experimental time-course characteristics of EGL against the N-acetylglucosamine pentamer substrate, (GlcNAc)(5), revealed higher production of (GlcNAc)(4) and lower production of (GlcNAc)(2) when compared with HEL. The saccharide-binding ability of subsites A-C in EGL was also found to be weaker than in HEL. An analysis of the enzymatic reactions of five mutants in respect of positions 34, 37 and 71 in HEL indicated the time-course characteristics of EGL to be caused by the combination of three substitutions (F34Y, N37G and G71R) between HEL and EGL. A computer simulation of the EGL-catalyzed reaction suggested that the time-course characteristics of EGL resulted from the difference in the binding free energy for subsites A, B, E and F and the rate constant of transglycosylation between EGL and HEL.     

Effects of yokukansan on behavioral and psychological symptoms of vascular dementia: an open-label trial

Previous clinical trials suggest that the traditional Japanese medicine yokukansan has beneficial effects on the behavioral and psychological symptoms of dementia (BPSD). The present study was conducted to elucidate the efficacy of yokukansan on BPSD in patients with vascular dementia. Thirteen Japanese patients (9 men and 4 women) who were diagnosed as having vascular dementia (VaD) according to the diagnostic criteria of NINDS-AIREN were subjected to the open-label clinical trial in which yokukansan (7.5g/day) has been given for 4 weeks. Their mean age was 71.2±6.5 years. The BPSD was evaluated using the Neuropsychiatric Inventory (NPI), cognitive function was evaluated by the Mini-Mental State Examination (MMSE), the activities of daily living was evaluated by Barthel index (BI) and Disability Assessment for Dementia (DAD), and the extrapyramidal signs were evaluated by United Parkinson's Disease Rating Scale (UPDRS). The mean NPI was 33.0±17.3 and 23.6±13.9 for the baseline and after treatment, respectively. It was significantly improved after treatment (p<0.05). In the NPI-subcategories, there was a significant improvement in agitation and disinhibition after the treatment. There was no significant change in MMSE, BI, DAD or UPDRS before and after the treatment. There was no adverse effect during the treatment period. The present results suggest that yokukansan is beneficial for the treatment of BPSD in VaD patients.