Gravity-induced modification of auxin transport and distribution for peg formation in cucumber seedlings: possible roles for CS-AUX1 and CS-PIN1

Cucurbit seedlings potentially develop a peg on each side of the transition zone between the hypocotyl and root. Seedlings grown in a horizontal position suppress the development of the peg on the upper side of the transition zone in response to gravity. It is suggested that this suppression occurs due to a reduction in auxin levels to below the threshold value. We show in this study that the free indole-3-acetic acid (IAA) content is low, while IAA conjugates are significantly more abundant in the upper side of the transition zone of gravistimulated seedlings, compared to the lower side. A transient increase in mRNA of the auxin-inducible gene, CS-IAA1, was observed in the excised transition zone. The result suggests that the transition zone is a source of auxin. Cucumber seedlings treated with auxin-transport inhibitors exhibited agravitropic growth and developed a peg on each side of the transition zone. Auxin-transport inhibitors additionally caused an increase in CS-IAA1 mRNA accumulation at the transition zone, indicating a rise in intracellular auxin concentrations due to a block of auxin efflux. To study the involvement of the auxin transport system in peg formation, we isolated the cDNAs of a putative auxin influx carrier, CS-AUX1, and putative efflux carrier, CS-PIN1, from cucumber (Cucumis sativus L.) plants. Both genes (CS-AUX1 in particular) were auxin-inducible. Accumulation of CS-AUX1 and CS-PIN1 mRNAs was observed in vascular tissue, cortex and epidermis of the transition zone. A reduced level of CS-AUX1 mRNA was observed in the upper side of the gravistimulated transition zone, compared with the lower side. It is therefore possible that a balance in the activities of auxin influx and efflux carriers controls intracellular auxin concentration at the transition zone, which results in lateral placement of a peg in cucumber seedlings.Abbreviations HFCA 9-hydroxyfluorene-9-carboxylic acid - IAA indole-3-acetic acid - NPA 1-N-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Vitamin D and bone

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.     

PEG-inhibitor conjugates: application of diphenyl alpha-aminoalkylphosphonate to removal of chymotrypsin

Diphenyl 1-amino-2-phenylethylphosphonate was introduced to poly(ethylene glycol)s (PEGs) with average molecular masses of 300, 400, and 600 to prepare water-insoluble PEG-inhibitor conjugates. Interestingly, only the conjugate from PEG with an average molecular weight of 600 formed a precipitate with chymotrypsin but not with trypsin. The results demonstrated that the PEG-inhibitor conjugate is useful for separation of chymotrypsin.     

FRL-1, a member of the EGF-CFC family,is essential for neural differentiation in Xenopus early development

Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. Here, we show that FRL-1, a Xenopus EGF-CFC gene, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induced the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppressed the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) showed that FRL-1 is required for neural development. FRL-1MO-injected embryos lacked neural structures but contained mesodermal tissue. It was suggested previously that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibited the expression of neural markers that were induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, we showed that the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response.     

Interaction between the antigen and antibody is controlled by the constant domains: normal mode dynamics of the HEL-HyHEL-10 complex

The antigen binding fragment (Fab) of a monoclonal antibody (HyHEL-10) consists of variable domains (Fv) and constant domains (CL-CH1). Normal modes have been calculated from the three-dimensional structures of hen egg lysozyme (HEL) with Fab, those of HEL with Fv, and so on. Only a small structural change was found between HEL-Fab and HEL-Fv complexes. However, HEL-Fv had a one order of magnitude lower dissociation constant than HEL-Fab. The Calpha fluctuations of HEL-Fab differed from those of HEL-Fv with normal mode calculation, and the dynamics can be thought to be related to the protein-protein interactions. CL-CH1 may have influence not only around local interfaces between CL-CH1 and Fv, but also around the interacting regions between HEL and Fv, which are longitudinally distant. Eighteen water molecules were found in HEL-Fv around the interface between HEL and Fv compared with one water molecule in HEL-Fab. These solvent molecules may occupy the holes and channels, which may occur due to imperfect complementarity of the complex. Therefore, the suppression of atomic vibration around the interface between Fv and HEL can be thought to be related to favorable and compact interface formation by complete desolvation. It is suggested that the ability to control the antigen-antibody affinity is obtained from modifying the CL-CH1. The second upper loop in the constant domain of the light chain (UL2-CL), which is a conserved gene in several light chains, showed the most remarkable fluctuation changes. UL2-CL could play an important role and could be attractive for modification in protein engineering.