Kruppel-like factor 4 attenuates osteoblast formation,function, and cross talk with osteoclasts

Osteoblasts not only control bone formation but also support osteoclast differentiation. Here we show the involvement of Kruppel-like factor 4 (KLF4) in the differentiation of osteoclasts and osteoblasts. KLF4 was down-regulated by 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in osteoblasts. Overexpression of KLF4 in osteoblasts attenuated 1,25(OH)₂D₃-induced osteoclast differentiation in co-culture of mouse bone marrow cells and osteoblasts through the down-regulation of receptor activator of nuclear factor κB ligand (RANKL) expression. Direct binding of KLF4 to the RANKL promoter repressed 1,25(OH)₂D₃-induced RANKL expression by preventing vitamin D receptor from binding to the RANKL promoter region. In contrast, ectopic overexpression of KLF4 in osteoblasts attenuated osteoblast differentiation and mineralization. KLF4 interacted directly with Runx2 and inhibited the expression of its target genes. Moreover, mice with conditional knockout of KLF4 in osteoblasts showed markedly increased bone mass caused by enhanced bone formation despite increased osteoclast activity. Thus, our data suggest that KLF4 controls bone homeostasis by negatively regulating both osteoclast and osteoblast differentiation.     

The Effect of Ageing on Ocular Blood Flow,Oxygen Tension and Retinal Function during and after Intraocular Pressure Elevation

Purpose

To investigate the effect of ageing on the recovery of ocular blood flow, intravitreal oxygen tension and retinal function during and after intraocular pressure (IOP) elevation.

Methods

Long Evans rats (3- and 14-month-old) underwent acute stepwise IOP elevation from 10 to 120 mmHg (5 mmHg steps each 3 minutes). IOP was then returned to baseline and recovery was monitored for 2 hours. Photopic electroretinograms (ERG) were recorded at each IOP step during stress and at each minute during recovery. Ocular blood flow and vitreal oxygen tension (pO₂) were assayed continuously and simultaneously using a combined laser Doppler flow meter (LDF) and an oxygen sensitive fibre-optic probe, respectively. The combined sensor was placed in the vitreous chamber, proximal to the retina. Data were binned into 3 minute intervals during stress and 1 min intervals during recovery. Recovery data was described using a bi-logistic function.

Results

Rats of both ages showed similar susceptibility to IOP elevation, with pO₂ showing a closer relationship to ERG than LDF. During recovery, both ages showed a distinctive two-phased recovery for all three measures with the exception of the LDF in 3-month-old rats, which showed only 1 phase. In all animals, LDF recovered fastest (<1 minute), followed by pO₂ (<10 minute) and ERG (>1 hour). 14-month-old rats showed surprisingly faster and greater LDF recovery compared to the younger group, with similar levels of pO₂ recovery. However, the ERG in these middle-aged animals did not fully recover after two hours, despite showing no difference in susceptibility to IOP during stress compared to the young group.

Conclusions

Young and middle-aged eyes showed similar susceptibility to IOP elevation in terms of pO₂, LDF and ERG. Despite this lack of difference during stress, older eyes did not completely recover function, suggesting a more subtle age-related susceptibility to IOP.     

Investigation of four candidate genes (IGF2, JHDM1A, COPB1 and TEF1) for growth rate and backfat thickness traits on SSC2q in Large White pigs

As important quantitative traits, the growth rate and backfat thickness are controlled by multiple genes. The aim of this investigation was to evaluate the effect of the single and multiple SNPs of four candidate genes (IGF2, JHDM1A, COPB1 and TEF-1) on growth rate and backfat thickness. The four candidate genes were mapped on the p arm of SSC 2, and there are several QTLs, such as average daily gain, backfat thickness, an imprinted QTLs affecting muscle mass and fat deposition have been reported in this region. The polymorphisms of these genes were detected using PCR–RFLP methods, mixed procedure was used to analyze the single marker association with the growth and backfat thickness traits, and the gene–gene combination was investigated using multiple-markers analysis. The single marker association analysis indicated that the IGF2 intron-3 g.3072G > A and the substitution g.93G > A of TEF-1 gene were significantly associated with the age at 100 kg (P < 0.05). The JHDM1A 3′UTR g.224C > G, the c.3096C > T polymorphism of COPB1 gene and the substitution g.93G > A of TEF-1 gene were all significantly associated with the backfat at the shoulder (P < 0.05), backfat at the last rib, backfat at the lumbar, and the average backfat thickness, respectively. The multiple-markers analysis indicated that IGF2 and TEF-1 integrated gene networks for the age at 100 kg. Therefore, we can suggest that the polymorphism of IGF2 and TEF-1 gene could be used in marker-assisted selection for the age at 100 kg in Large White pigs.     

Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold,Cep192, in the Regulation of Bipolar Spindle Formation

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study with Xenopus laevis egg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif of Xenopus Cep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous to Xenopus p-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.     

Ultrastructural analysis of ineffective alfalfa nodules formed by nif::Tn5 mutants of Rhizobium meliloti.

Ineffective alfalfa nodules formed by Rhizobium meliloti nif::Tn5 mutants were examined by light and electron microscopy. R. meliloti nifH::Tn5 mutants formed nodules that were similar in structure to wild-type nodules except that nifH- bacteroids accumulated a compact, electron-dense body. In contrast to nodules induced by wild type and nifH mutants, nifDK- R. meliloti mutants induced nodules which contained numerous starch grains and prematurely senescent bacteroids. In addition, meristematic activity in nifDK- nodules ceased significantly earlier than in nifH- nodules. All mutant nodules exhibited elevated levels of rough endoplasmic reticulum and Golgi membranes compared to wild-type nodule cells. These elevated levels may reflect either a response to nitrogen starvation in the ineffective nodules or an abnormal synthesis and export of nodule-specific proteins during later developmental stages.     

Identification and characterization of a novel angiostatin-binding protein by the display cloning method

Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of 3.4 x 10(-7) M. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.     

Physical mapping and microsynteny of Brassica rapa ssp. pekinensis genome corresponding to a 222 kbp gene-rich region of Arabidopsis chromosome 4 and partially duplicated on chromosome 5

We constructed a bacterial artificial chromosome (BAC) library, designated as KBrH, from high molecular weight genomic DNA of Brassica rapa ssp. pekinensis (Chinese cabbage). This library, which was constructed using HindIII-cleaved genomic DNA, consists of 56,592 clones with average insert size of 115 kbp. Using a partially duplicated DNA sequence of Arabidopsis, represented by 19 and 9 predicted genes on chromosome 4 and 5, respectively, and BAC clones from the KBrH library, we studied conservation and microsynteny corresponding to the Arabidopsis regions in B. rapa ssp. pekinensis. The BAC contigs assembled according to the Arabidopsis homoeologues revealed triplication and rearrangements in the Chinese cabbage. In general, collinearity of genes in the paralogous segments was maintained, but gene contents were highly variable with interstitial losses. We also used representative BAC clones, from the assembled contigs, as probes and hybridized them on mitotic (metaphase) and/or meiotic (leptotene/pachytene/metaphase I) chromosomes of Chinese cabbage using bicolor fluorescence in situ hybridization. The hybridization pattern physically identified the paralogous segments of the Arabidopsis homoeologues on B. rapa ssp. pekinensis chromosomes. The homoeologous segments corresponding to chromosome 4 of Arabidopsis were located on chromosomes 2, 8 and 7, whereas those of chromosome 5 were present on chromosomes 6, 1 and 4 of B. rapa ssp. pekinensis.     

Genome constitutions of Roegneriaalashanica,R. elytrigioides,R. magnicaespes and R. grandis (Poaceae: Triticeae) via genomic in‐situ hybridization

The genomic constitutions of Roegneria alashanica, R. elytrigioides, R. magnicaespes and R. grandis were studied using GISH. DNA of Pseudoroegneria spicata (St), P. libanotica (St), P. stipifolia (St), R. ciliaris (StY), Lopophyllum elongatum (E^e), Agropyron cristatum (P) and Hordeum bogdanii (H) were used for probing, respectively. The results indicated that: 1) R. alashanica and R. magnicaespes contained one St genome, the other genome was unidentified, however, it was not an E, P, H or Y genome; 2) R. elytrigioides contained two St genomes and should on this basis be included in Pseudoroegneria; 3) R. grandis contained an St and an St^g genome. The St^g genome is suggested to be a modified form of the St genome of Pseudoroegneria and to be homoeologous with the Y genome. It might be an intermediate type between the St and Y genomes. Therefore, R. elytrigioides should be treated as Pseudoroegneriaelytrigioides.Roegneria alashanica, R. magnicaespes and R. grandis does apparently not belong in the genus Roegneria but further studies are needed to establish their correct taxonomic position.