A knee-specific finite element analysis of the human anterior cruciate ligament impingement against the femoral intercondylar notch

This work presents a finite element analysis of anterior cruciate ligament (ACL) impingement against the intercondylar notch during tibial external rotation and abduction, as a mechanism of noncontact ACL injuries. Experimentally, ACL impingement was measured in a cadaveric knee in terms of impingement contact pressure and six degrees-of-freedom tibiofemoral kinematics. Three-dimensional geometries of the ACL, femur and tibia were incorporated into the finite element model of the individual knee specimen. A fiber-reinforced model was adopted, which accounts for the anisotropy, large deformation, nonlinearity and incompressibility of the ACL. With boundary conditions specified based on the experimental tibiofemoral kinematics, the finite element analysis showed that impingement between the ligament and the lateral wall of intercondylar notch could occur when qthe knee at 45° was externally rotated at 29.1° and abducted at 10.0°. Strong contact pressure and tensile stress occurred at the impinging and nonimpinging sides of the ligament, respectively. The impingement force and contact area estimated from the model matched their counterparts from the corresponding cadaver experiment. The modeling and experimental approach provides a useful tool to characterize potential ACL impingement on a knee-specific basis, which may help elucidate the ACL injury mechanism and develop more effective treatments.     

Homeoprotein Hbx4 represses the expression of the adhesion molecule DdCAD-1 governing cytokinesis and development

We investigated the function of homeodomain-containing protein Hbx4 in Dictyostelium discoideum. Hbx4-overexpressing cells (Hbx4(OE)) displayed defects in growth rate and cytokinesis and showed differences in slug motility and cell-type proportioning from KAx3. Furthermore, the overexpression of Hbx4 inhibited the induction of cadA, which encoded the Ca(2+)-dependent cell adhesion molecule DdCAD-1, despite expression of csaA and gpaB. The electrophoretic mobility shift assay showed that the promoter of cadA contained the Hbx4-binding site. Moreover, constitutively expressed DdCAD-1 in Hbx4(OE) rescued the defects in cytokinesis and development. These results suggest that Hbx4 modulates DdCAD-1-mediated cytokinesis and cell-type proportioning.     

α-Poly-l-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes

α-Poly-l-lysine (PLL) has been used for various purposes such as cell attachment, immunization, and molecular delivery, and is known to be cytotoxic to several cell lines. Here, we studied the effect of PLL on the adipogenesis of 3T3-L1 cells and investigated the underlying mechanism. Differentiation media containing PLL with a molecular weight (MW) greater than 4 kDa enhanced lipid droplet formation and increased adipogenic marker levels, indicating an increase in adipocyte differentiation. PLL with a molecular weight between 30 and 70 kDa was more effective than PLL of other sizes in 3T3-L1 cell differentiation. Moreover, PLL induced 3T3-L1 adipogenesis in insulin-free adipocyte differentiation medium. Incubation with insulin and PLL exhibited greater adipogenesis than insulin treatment only even at a high concentration. PLL stimulated insulin signaling and augmented the signaling pathway when it was added with insulin. While PLL did not activate the glucocorticoid receptor, which is phosphorylated by dexamethasone (DEX), it showed a positive effect on the cAMP signal pathway when preadipocytes were treated with PLL and 3-isobutyl-1-methylxanthine (IBMX). Consistent with these results, incubation with PLL and DEX without IBMX induced adipocyte differentiation. We also observed that the mitotic clonal expansion phase was the critical stage in adipogenesis for inducing the effects of PLL. These results suggest that PLL functions as an adipogenic inducer in 3T3-L1 preadipocytes and PLL has a direct effect on insulin signaling, one of the main regulatory pathways.

     

Reduced glutathione levels affect the culmination and cell fate decision in Dictyostelium discoideum

Glutaredoxins have been known to be glutathione-dependent oxidoreductases that participate in the redox regulation of various cellular processes. To understand the role of glutaredoxins in the development, we examined glutaredoxin 1 (Grx1) of Dictyostelium discoideum. Its mRNA was highly accumulated at the mound and the culmination stages. When Grx1-overexpressing cells were developed, their culmination was delayed, and the expression of marker genes for prespore and spore decreased. Interestingly, they had about 1.5-fold higher amount of reduced glutathione (GSH) compared with parental cells and their prolonged migration was repressed by the oxidant such as hydrogen peroxide. To confirm the effect of GSH on the culmination, glutathione reductase (Gsr) was overexpressed or underexpressed. Similar to Grx1-overexpressing cells, Gsr-overexpressing cells contained about 1.5-fold higher amount of GSH and exhibited the delayed culmination. In contrast, the knockdown mutant of Gsr had nearly 50% lower amount of GSH and showed accelerated culmination. Taken together, these data suggest that the culmination of Dictyostelium is controlled by GSH. In addition, the cells having higher GSH levels showed a prestalk tendency in the chimeric slugs with parental cells, indicating that the difference in the amount of GSH may affect the determination of cell fate.     

An AFLP-based linkage map of Japanese red pine (Pinus densiflora) using haploid DNA samples of megagametophytes from a single maternal tree

We have constructed an AFLP-based linkage map of Japanese red pine (Pinus densiflora Siebold et Zucc.) using haploid DNA samples of 96 megagametophytes from a single maternal tree, selection clone Kyungbuk 4. Twenty-eight primer pairs generated a total of 5,780 AFLP fragments. Five hundreds and thirteen fragments were verified as genetic markers with two alleles by their Mendelian segregation. At the linkage criteria LOD 4.0 and maximum recombination fraction 0.25(theta), a total of 152 markers constituted 25 framework maps for 19 major linkage groups. The maps spanned a total length of 2,341 cM with an average framework marker spacing of 18.4 cM. The estimated genome size was 2,662 cM. With an assumption of equal marker density, 82.2% of the estimated genome would be within 10 cM of one of the 230 linked markers, and 68.1% would be within 10 cM of one of the 152 framework markers. We evaluated map completeness in terms of LOD value, marker density, genome length, and map coverage. The resulting map will provide crucial information for future genomic studies of the Japanese red pine, in particular for QTL mapping of economically important breeding target traits.     

Glutathione is required for growth and prespore cell differentiation in Dictyostelium

Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells and acts as reducing equivalent in many cellular processes. We investigated the role of glutathione in Dictyostelium development by disruption of gamma-glutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis. GCS-null strain showed glutathione auxotrophy and could not grow in medium containing other thiol compounds. The developmental progress of GCS-null strain was determined by GSH concentration contained in preincubated media before development. GCS-null strain preincubated with 0.2 mM GSH was arrested at mound stage or formed bent stalk-like structure during development. GCS-null strain preincubated with more than 0.5 mM GSH formed fruiting body with spores, but spore viability was significantly reduced. In GCS-null strain precultured with 0.2 mM GSH, prestalk-specific gene expression was delayed, while prespore-specific gene and spore-specific gene expressions were not detected. In addition, GCS-null strain precultured with 0.2 mM GSH showed prestalk tendency and extended G1 phase of cell cycle. Since G1 phase cells at starvation differentiate into prestalk cells, developmental defect of GCS-null strain precultured with 0.2 mM GSH may result from altered cell cycle. These results suggest that glutathione itself is essential for growth and differentiation to prespore in Dictyostelium.     

Calcium-induced conformational changes of the recombinant CBP3 protein from Dictyostelium discoideum

Calcium-binding proteins play various and significant roles in biological systems. Conformational changes in their structures are closely related to their physiological functions. To understand the role of calcium-binding protein 3 (CBP3) in Dictyostelium discoideum, its recombinant proteins were analyzed using circular dichroism (CD) and fluorescence spectroscopy. Gel mobility shift analysis showed that Ca2+ induced a mobility shift of the recombinant CBP3. Far ultra-violet CD spectra and intrinsic fluorescence spectra on CBP3 and its N- and C-terminal domains exhibited that they underwent a conformational rearrangement depending upon Ca2+ binding. Measurement of Ca2+ dissociation constants demonstrated that CBP3 had high affinity toward Ca2+ in the sub-micromolar range and N-terminal domain had higher affinity than C-terminal domain. The changes of fluorescence spectra by an addition of 8-anilino-1-naphthalene sulfonic acid indicated that the hydrophobic patches of CBP3 and its C-terminal domain are likely to be more exposed in the presence of Ca2+. Since the exposure of hydrophobic patches is thermodynamically unfavorable, Ca2+-bound CBP3 may interact with other proteins in vivo. All these data suggest that Ca2+ induces CBP3 to be more favorable conformation to interact with target proteins.     

Crystallization and preliminary X-ray crystallographic analysis of the pediocin immunity protein (PedB) from Pediococcus pentosaceus at 1.35 A resolution

PedB, a bacterial immunity protein conferring immunity to a newly identified pediocin (pediocin PP-1), was crystallized by the hanging-drop vapor diffusion method at 296 K. A 1.35 A data set has been collected from a single crystal at 100 K using synchrotron-radiation source. The PedB crystals belong to the hexagonal space group P6(2) or P6(4), with unit cell parameters a = b = 62.2, c = 39.9 A. Analysis of the packing density shows that the asymmetric unit probably contains one molecule with a solvent content of 33.8%.     

High resolution crystal structure of PedB: a structural basis for the classification of pediocin-like immunity proteins

Background  

Pediocin-like bacteriocins, ribosomally-synthesized antimicrobial peptides, are generally coexpressed with cognate immunity proteins in order to protect the bacteriocin-producer from its own bacteriocin. As a step for understanding the mode of action of immunity proteins, we determined the crystal structure of PedB, a pediocin-like immunity protein conferring immunity to pediocin PP-1.