Normal-phase HPLC quantitation of chlorophyll a′ and phylloquinone in Photosystem I particles

Fractionated Photosystem (PS) I particles consisting of six, five or two core proteins were analyzed by HPLC for chlorophyll (Chl) a and phylloquinone (PhQ). Each particle had a Chl a/P700 molar ratio of 50–55 and contained ca. 2 molecules of Chl a per P700. Deliberate control of eluent composition led to isolated elution of PhQ and -carotene in the normal-phase chromatogram. Based on these a simple HPLC procedure has been established to determine the PhQ/P700 molar ratio, which was ca. 2 for the larger two PS I particles and ca. 1 for the smallest particle, in line with previous reports.     

Possibility of selection of chondrogenic progenitor cells by telomere length in FGF-2-expanded mesenchymal stromal cells

Telomere length plays an important role in regulating the proliferative capacity of cells, and serves as a marker for cell cycle history and also for their remaining replicative potential. Mesenchymal stromal cells (MSC) are known to be a significant cell source for therapeutic intervention and tissue engineering. To investigate any possible limitations in the replicative potential and chondrogenic differentiation potential of fibroblast growth factor-2-expanded MSCs (FGF(+)MSC), these cells were differentiated at various population doublings (PDs), and telomere length and telomerase activity were measured before and after differentiation. FGF(+)MSC cultured at a relatively low density maintained proliferation capability past more than 80 PD and maintained chondrogenic differentiation potential up to at least 46 PD and long telomeres up to 105 PD, despite expressing low levels of telomerase activity. Interestingly, upon chondrogenic differentiation of these cells, telomeres showed a remarkable reduction in length. This shortening was more extensive when FGF(+)MSC of higher PD levels were differentiated. These findings suggest that telomere length may be a useful genetic marker for chondrogenic progenitor cells.     

CD8+ T cells play disparate roles in the induction and the effector phases of murine experimental allergic conjunctivitis

Although CD4+ Th2 cells clearly play an essential role in the development of experimental allergic diseases, the functions CD8+ T cells may have in these diseases have been investigated less extensively and remain controversial. Here, we investigated the roles of CD8+ T cells in the development of experimental allergic conjunctivitis (EC). EC was induced in CD8alpha-deficient (CD8KO) mice and wild-type (WT) mice by active immunization with short ragweed pollen (RW) followed by challenge with RW-containing eye drops. Alternatively, EC was induced by transferring RW-primed splenocytes followed by RW challenge. With regard to actively immunized mice, CD8KO mice showed significantly less severe eosinophil infiltration of the conjunctiva and lower total IgE levels, although the levels of the other Igs were equivalent between the two strains. Cytokine production by cultured splenocytes also did not differ, but the WT conjunctivas showed upregulated IL-5 and IL-6 expression and greater upregulation of IL-4 expression than the conjunctivas of CD8KO mice. Thus, CD8+ T cells may play a significant role during the induction phase by aiding IgE production and the generation of Th2 cytokines in the conjunctiva, thus promoting the development of EC. In contrast, splenocytes from CD8KO mice induced significantly more severe EC in WT mice than cells from WT mice. In addition, transfer of RW-primed splenocytes induced significantly more severe eosinophil infiltration in CD8KO recipient mice. Thus, CD8+ T cells promote the development of EC during the induction phase, but suppress it during the effector phase.     

Interleukin-1β-Induced Autophagy-Related Gene 5 Regulates Proliferation of Embryonic Stem Cell-Derived Odontoblastic Cells

We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg) 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells.     

NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits. hLH alpha (N-glycosylated at Asn52 and Asn78) and hLH beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH beta and from glycopeptides obtained after tryptic digestion of hLH alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) 1H-NMR spectroscopy. The results indicate predominantly diantennary. N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH alpha) and Asn30 (hLH beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha 1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc alpha 2-6 GalNAc beta 1-4GlcNAc beta 1-2 Man alpha 1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [formula, see text]     

Changes in the amount of the diapause factor in the subesophageal ganglion during development of the silkworm, Bombyx mori

Changes in the diapause factor content of the subesophageal ganglion of the silkworm were examined from the viewpoint of sex and voltinism differences. The diapause factor content was comparatively low from the 4th larval ecdysis to the larval-pupal ecdysis, irrespective of sex and voltinism. However, remarkable changes occurred after the larval-pupal ecdysis. An interesting finding is that the diapause factor content in the female diapause egg-producer decreased gradually with pupal-adult development, but in the male diapause egg-producer as well as in the non-diapause egg-producer it increased up to the half-way stage in the pupal-adult development. This suggests that the diapause factor is not secreted from the subesophageal ganglion of the male pharate adult of the diapause egg-producer. This finding was confirmed by implantation experiments using brain-subesophageal ganglion complex or only subesophageal ganglion.     

A new EPR signal associated with photosystem 1 of algal and plant photosynthesis

A new low temperature electron paramagnetic resonance (EPR) signal with a g-value of 1.97 was found in Photosystem-1 particles from a blue-green alga, Anacystis nidulans, illuminated at room temperature. A similar signal was also found in spinach Photosystem-1 particles treated with thiophenol to decrease interference from a signal due to Center A. In the dark, the signal appeared only when the Anacystis particles were at redox potentials lower than -0.5 volts where Centers A and B were also reduced. The signal is most likely due to another iron-sulfur cluster, tentatively designated as Center C. Center C could be photoreduced at low temperatures like Center A when Centers A and B were partially reduced prior to illumination, indicating possible close association of these centers in Photosystem 1 of green plant and algal photosynthesis.     

Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).     

Numerical prediction of a relation among growth,reproduction and mortality in iteroparous fish populations

Summary As a quantitative approach to the life history, the present paper numerically analyzed a relation among growth, reproduction and mortality for nine fish populations with a technique of optimal control theory, the discrete maximum principle. The analytical method used was derived on the postulate that natural selection maximized the net reproductive rate subject to a few constraints. A comparison between the theoretical and observed survival rate showed that the former was discernibly lower than the latter in all the populations except the three species. For the reasons mentioned below, however, the analyzed life history data should not be interpreted as a refutation of the adopted postulate and the method. First, it is generally very difficult to obtain a good estimate of the rate with traditional methods. Moreover, it is probable in most fish populations that the rate considerably changes with age even in the adult stage though it is usually estimated on the assumption that it is constant in a certain age range. Second, an intense fishing pressure possibly alters the life history characteristics of fish populations to some extent.