YCZ-18 Is a New Brassinosteroid Biosynthesis Inhibitor

Plant hormone brassinosteroids (BRs) are a group of polyhydroxylated steroids that play critical roles in regulating broad aspects of plant growth and development. The structural diversity of BRs is generated by the action of several groups of P450s. Brassinazole is a specific inhibitor of C-22 hydroxylase (CYP90B1) in BR biosynthesis, and the application use of brassinazole has emerged as an effective way of complementing BR-deficient mutants to elucidate the functions of BRs. In this article, we report a new triazole-type BR biosynthesis inhibitor, YCZ-18. Quantitative analysis the endogenous levels of BRs in Arabidopsis indicated that YCZ-18 significantly decreased the BR contents in plant tissues. Assessment of the binding affinity of YCZ-18to purified recombinant CYP90D1 indicated that YCZ-18 induced a typical type II binding spectrum with a K_d value of approximately 0.79 μM. Analysis of the mechanisms underlying the dwarf phenotype associated with YCZ-18 treatment of Arabidopsis indicated that the chemically induced dwarf phenotype was caused by a failure of cell elongation. Moreover, dissecting the effect of YCZ-18 on the induction or down regulation of genes responsive to BRs indicated that YCZ-18 regulated the expression of genes responsible for BRs deficiency in Arabidopsis. These findings indicate that YCZ-18 is a potent BR biosynthesis inhibitor and has a new target site, C23-hydroxylation in BR biosynthesis. Application of YCZ-18 will be a good starting point for further elucidation of the detailed mechanism of BR biosynthesis and its regulation.     

Wider Retinal Artery Trajectories in Eyes with Macular Hole Than in Fellow Eyes of Patients with Unilateral Idiopathic Macular Hole

Purpose

To determine whether the width of the retinal artery (RA) trajectory was associated with the presence of a macular hole (MH).

Methods

A retrospective cross sectional case-control study was performed. The fundus photographs were rotated 90 degrees, and the coordinates of the best fit curve of the RA trajectory were determined automatically based on these plots using the ImageJ program. The converted coordinates were fit to a second degree polynomial (ax²/100 + bx + c) equation. The width and steepness of the RA trajectory, “a”, of the eyes with a MH eye were compared to that of the fellow eyes.

Results

One hundred and ten eyes of 55 consecutive patients (30 women) with a unilateral MH and healthy fellow eyes were analyzed. The mean age was 64.9 years (range 47-81 years). The constant ‘a’ was significantly smaller in eyes with a MH than that of the fellow eyes (0.379 ± 0.094 vs 0.416 ± 0.121, P = 0.001, paired t test), indicating that the RA trajectory was wider in the MH eyes than in the fellow eyes. There was a significant correlation between the axial length and ‘a’ of the RA trajectory in the MH eyes (R = 0.273, P = 0.044) and in the fellow eyes (R = 0.356, P = 0.008; Spearman’s rank correlation coefficient).

Conclusions

Because eyes with a MH have a significantly wider and flatter RA trajectory, there may be greater traction on the fovea which is located between the RA arches. The causative role of this finding is still unclear.     

Bovine alpha-2-HS-glycoprotein functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and SIVmac239 envelope glycoprotein

The presence of anti-CCR5 and anti-HIV-1 envelope glycoprotein (ENV) gp41 antibodies (Abs) at sites of HIV-1 exposure was effective in preventing its transmission to HIV-1-exposed seronegative (ESN) subjects. Here, we design an immunogen that can induce Abs against CCR5 and SIVmac239 ENV simultaneously and show that bovine alpha-2-HS-glycoprotein (bAHSG) functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and ENV. Initially, we generated a rhesus CCR5-derived cyclopeptide (cDDR5) conjugated with a recombinant trimeric SIVmac239 Env. When inguinally administered to rhesus macaques, the immunogen simultaneously induced both anti-CCR5 and anti-ENV Abs in sera, and the purified serum IgG fraction exerted an inhibitory effect on SIVmac239 infection in vitro. When further boosted with bAHSG, the responses of both Abs were significantly enhanced. To examine the cross-reactivity of bAHSG, it was administered to naïve cynomolgus macaques. The results showed a statistically significant increase in IgG response against cynomolgus CCR5 and SIVmac239 ENV, and the induction of neutralizing activity against SIVmac239. These findings suggest that bAHSG is useful for immune strategies aimed at generating Abs against CCR5 and ENV simultaneously to confer HIV-protective immunity.     

Flowering and Endogenous Levels of Plant Hormones in Lemna Species

The occurrence and endogenous level of various plant hormoneswere measured for the short-day plants Lemna paucicostata 151and 381 and the long-day plant Lemna gibba G3 to determine whetherany of them are involved in the photoperiodic control of flowering.ABA, IAA, GA₁, GA₂₉, GA₃₄, GA₅₃, trans- and cis-zeatin, trans-and cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenine and N⁶-(²-isopentenyl)adenosine were definitely detected in each species, while GA₄was only detected in L. gibba G3 and GA₂₀ was only detectedin L. paucicostata 151. The endogenous levels of ABA and IAAwere in the range of 1–7 ng/g fr wt and were not significantlydifferent in vegetative and flowering plants. The endogenousgibberellin levels were generally higher in Lemna grown underlong-day rather than short-day conditions. The endogenous cytokininlevels were almost the same in both flowering and vegetativeplants of L. paucicostata 151 and 381. In L. gibba G3, however,the level of cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenineand N⁶-(²-sopentenyl) adenosine were higher in vegetative thanin flowering plants. These results indicate that there is not necessarily a directrelation between endogenous plant hormone levels and flowering,and that the chemical basis for the photoperiodic control offlowering cannot be explained solely by changes in hormone levels.The possibility remains, however, that one or more of the planthormones has some influence of secondary importance on the floweringprocess in Lemna. (Received January 29, 1986; Accepted July 12, 1986)     

Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice

The factors controlling the migration of mammalian gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus are not well understood. We studied whether the extracellular calcium-sensing receptor (CaR) promotes migration/chemotaxis of GnRH neurons. We demonstrated expression of CaR in GnRH neurons in the murine basal forebrain and in two GnRH neuronal cell lines: GT1-7 (hypothalamus derived) and GN11 (olfactory bulb derived). Elevated extracellular Ca(2+) concentrations promoted chemotaxis of both cell types, with a greater effect in GN11 cells. This effect was CaR mediated, as, in both cell types, overexpression of a dominant-negative CaR attenuated high Ca(2+)-stimulated chemotaxis. We also demonstrated expression of a beta-chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CC motif receptor-2 (CCR2), in the hypothalamic GnRH neurons as well as in GT1-7 and GN11 cells. Exogenous MCP-1 stimulated chemotaxis of both cell lines in a dose-dependent fashion; the effect was greater in GN11 than in GT1-7 cells, consistent with the higher CCR2 mRNA levels in GN11 cells. Activating the CaR stimulated MCP-1 secretion in GT1-7 but not in GN11 cells. MCP-1 secreted in response to CaR stimulation is biologically active, as conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of GN11 cells, and this effect was partially attenuated by a neutralizing antibody to MCP-1. Finally, in the preoptic area of anterior hypothalamus, the number of GnRH neurons was approximately 27% lower in CaR-null mice than in mice expressing the CaR gene. We conclude that the CaR may be a novel regulator of GnRH neuronal migration likely involving, in part, MCP-1.     

Macrophage inflammatory protein-1α (MIP-1α) enhances a receptor activator of nuclear factor κB ligand (RANKL) expression in mouse bone marrow stromal cells and osteoblasts through MAPK and PI3K/Akt pathways

Osteolytic lesions are rapidly progressive during the terminal stages of myeloma, and the bone pain or bone fracture that occurs at these lesions decreases the patients’ quality of life to a notable degree. In relation to the etiology of this bone destruction, it has been reported recently that MIP-1α, produced in large amounts in myeloma patients, acts indirectly on osteoclastic precursor cells, and activates osteoclasts by way of bone-marrow stromal cells or osteoblasts, although the details of this process remain obscure. In the present study, our group investigated the mechanism by which RANKL expression is induced by MIP-1α and the effects of MIP-1α on the activation of osteoclasts. RANKL mRNA and RANKL protein expressions increased in both ST2 cells and MC3T3–E1 cells in a MIP-1α concentration-dependent manner. RANKL mRNA expression began to increase at 1 h after the addition of MIP-1α; the increase became remarkable at 2 h, and continuous expression was observed subsequently. Both ST2 and MC3T3-E1 cells showed similar levels of increased RANKL protein expression at 1, 2, and 3 days after the addition of MIP-1α. After the addition of MIP-1α, the amount of phosphorylated ERK1/2 and Akt protein expressions showed an increase, as compared to the corresponding amount in the control group. On the other hand, the amount of phosphorylated p38MAPK protein expression showed a decrease from the amount in the control group after the addition of MIP-1α. U0126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor) was added to ST2 and MC3T3-E1 cells, and was found to inhibit RANKL mRNA and RANKL protein expression in these cells. When SB203580, a p38MAPK inhibitor, was added, RANKL mRNA and RANKL protein expression were increased in these cells. MIP-1α was found to promote osteoclastic differentiation of C7 cells, an osteoclastic precursor cell line, in a MIP-1α concentration-dependent manner. MIP-1α promoted differentiation into osteoclasts more extensively in C7 cells incubated together with ST2 and MC3T3-E1 cells than in C7 cells incubated alone. These results suggested that MIP-1α directly acts on the osteoclastic precursor cells and induces osteoclastic differentiation. This substance also indirectly induces osteoclastic differentiation through the promotion of RANKL expression in bone-marrow stromal cells and osteoblasts. The findings of this investigation suggested that activation of the MEK/ERK and the PI3K/Akt pathways and inhibition of p38MAPK pathway were involved in RANKL expression induced by MIP-1α in bone-marrow stromal cells and osteoblasts. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.     

Nematicidal activity of 5-hydroxymethyl-2-furoic acid against plant-parasitic nematodes

A nematicide, 5-hydroxymethyl-2-furoic acid (1), was isolated from cultures of the fungus Aspergillus sp. and its structure was identified by spectroscopic analysis. Compound 1 showed effective nematicidal activities against the pine wood nematode Bursaphelenchus xylophilus and the free-living nematode Caenorhabditis elegans without inhibitory activity against plant growth, but 1 did not show any effective nematicidal activity against Pratylenchus penetrans.     

Delimitation of cryptic species of the Scytosiphon lomentaria complex (Scytosiphonaceae,Phaeophyceae) in Japan,based on mitochondrial and nuclear molecular markers

Scytosiphon lomentaria (Scytosiphonaceae, Ectocarpales) is believed to include some cryptic species, particularly in the Pacific. We attempted to delimit these species in Japan using mitochondrial cox1 and cox3 and nuclear ITS2 and the second intron of the centrin gene (cetn‐int2). Fifty‐three cox1+cox3 mitotypes, 26 ITS2 ribotypes and 45 cetn‐int2 haplotypes were found in 107 samples collected from 33 localities in Japan. Based on phylogenetic analyses, similar sequence types were grouped into ten mitogroups, eight ribogroups and six cetn‐int2 haplogroups (sequence‐type groups). From the molecular trees and combinations of the mito‐, ribo‐ and haplogroups, three cryptic species were apparent (Groups I–III). Group I, widely distributed on Pacific coasts, was highly supported by all molecular trees, whereas Groups II (North Pacific) and III (Northwestern Pacific and Australasia) were more closely related to each other. However, sequence‐type‐group combinations that would be characteristic of hybrids between Groups II and III were not detected, suggesting no gene flow between the two Groups. Further investigations of an additional 127 sympatrically growing plants supported the absence of gene flow between Groups II and III. Four samples did not belong to any of the Groups I–III and possibly represent additional species.