Establishment and characterization of cultured epithelial cells lacking expression of ZO-1

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.     

SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice

We analyzed recessive mutants of two homeotic genes in rice, SUPERWOMAN1 (SPW1) and DROOPING LEAF (DL). The homeotic mutation spw1 transforms stamens and lodicules into carpels and palea-like organs, respectively. Two spw1 alleles, spw1-1 and spw1-2, show the same floral phenotype and did not affect vegetative development. We show that SPW1 is a rice APETALA3 homolog, OsMADS16. In contrast, two strong alleles of the dl locus, drooping leaf-superman1 (dl-sup1) and drooping leaf-superman2 (dl-sup2), cause the complete transformation of the gynoecium into stamens. In these strong mutants, many ectopic stamens are formed in the region where the gynoecium is produced in the wild-type flower and they are arranged in a non-whorled, alternate pattern. The intermediate allele dl-1 (T65), results in an increase in the number of stamens and stigmas, and carpels occasionally show staminoid characteristics. In the weakest mutant, dl-2, most of the flowers are normal. All four dl alleles cause midrib-less drooping leaves. The flower of the double mutant, spw1 dl-sup, produces incompletely differentiated organs indefinitely after palea-like organs are produced in the position where lodicules are formed in the wild-type flower. These incompletely differentiated organs are neither stamens nor carpels, but have partial floral identity. Based on genetic and molecular results, we postulate a model of stamen and carpel specification in rice, with DL as a novel gene controlling carpel identity and acting mutually and antagonistically to the class B gene, SPW1.     

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.     

pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture

Controlling cell proliferation during cell culturing is an effective way to improve the production yield in mammalian cell culture. We examined the effect of temperature shifts (TS) under pH control conditions in Chinese hamster ovary cells. When we shifted the culture temperature from 37 °C to 31 °C before a stationary phase at pH 6.8 (TS/pH 6.8), cell viability remained high, and the final human monoclonal antibody (hMab) concentration increased to 2.3 times that in the culture remaining at 37 °C. However, there were no significant effects on the cell viability or production yield with the same TS at pH 7.0 (TS/pH 7.0). The average specific hMab productivity and mRNA level of TS/pH 7.0 were the same as that of TS/pH 6.8. The control of cell growth by the TS or the addition of rapamycin was effective in the maintenance of cell viability, but there was no significant increase of the average specific hMab productivity in the culture where cell proliferation was controlled with rapamycin. The hMab mRNA concentration decreased to 55%–65% at a 37 °C culture with the addition of actinomycin D. In contrast, actinomycin D did not affect the mRNA level in the TS culture. This result suggested that the increase in the mRNA level in the TS condition was caused by an increase in mRNA stability. In this study, we show that TS can produce two unrelated effects: a prolongation of cell longevity and an improvement in mRNA stability.     

Prolamellar bodies formed by cyanobacterial protochlorophyllide oxidoreductase in Arabidopsis

In angiosperms, chlorophyll biosynthesis is light dependent. A key factor in this process is protochlorophyllide oxidoreductase (POR), which requires light to catalyze the reduction of protochlorophyllide to chlorophyllide. It is believed that this protein originated from an ancient cyanobacterial enzyme that was introduced into proto‐plant cells during the primary symbiosis. Here we report that PORs from the cyanobacteria Gloeobacter violaceus PCC7421 and Synechocystis sp. PCC6803 function in plastids. First, we found that the G. violaceus POR shows a higher affinity to its substrate protochlorophyllide than the Synechocystis POR but a similar affinity to plant PORs. Secondly, the reduced size of prolamellar bodies caused by a knockdown mutation of one of the POR genes, PORA, in Arabidopsis could be complemented by heterologous expression of the cyanobacterial PORs. Photoactive protochlorophyllide in the etioplasts of the complementing lines, however, was retained at a low level as in the parent PORA knockdown mutant, indicating that the observed formation of prolamellar bodies was irrelevant to the assembly of photoactive protochlorophyllide. This work reveals a new view on the formation of prolamellar bodies and provides new clues about the function of POR in the etioplast–chloroplast transition.     

Regulation of neuropeptide expression in the brain by neurotrophins

Neurotrophins, which are structurally related to nerve growth factor, have been shown to promote survival of various neurons. Recently, we found a novel activity of a neurotrophin in the brain: Brain-derived neurotrophic factor (BDNF) enhances expression of various neuropeptides. The neuropeptide differentiation activity was then compared among neurotrophins both in vivo and in vitro. In cultured neocortical neurons, BDNF and neurotrophin-5 (NT-5) remarkably increased levels of neuropeptide Y and somatostatin, and neurotrophin-3 (NT-3) also increased these peptides but required higher concentrations. At elevating substance P, however, NT-3 was as potent as BDNF. In contrast, NGF had negligible or no effect. Neurotrophins administered into neonatal brain exhibited slightly different potencies for increasing these neuropeptides: The most marked increase in neuropeptide Y levels was obtained in the neocortex by NT-5, whereas in the striatum and hippocampus by BDNF, although all three neurotrophins increased somatostatin similarly in all the brain regions examined. Overall spatial patterns of the neuropeptide induction were similar among the neurotrophins. Neurons in adult rat brain can also react with the neurotrophins and alter neuropeptide expression in a slightly different fashion. Excitatory neuronal activity and hormones are known to change expression of neurotrophins. Therefore, neurotrophins, neuronal activity, and hormones influence each other and all regulate neurotransmitter/peptide expression in developing and mature brain. Physiological implication of the neurotransmitter/peptide differentiation activities is also discussed.     

Genetic diversity and structure of the tropical seagrass Cymodocea serrulata spanning its central diversity hotspot and range edge

Aquatic Ecology - Persistence of populations at their distributional ranges relies on local population dynamics and the fitness of species with low dispersal potential. We analyzed the population...     

The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

 We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.     

Anti-microtubule ‘plinabulin’ chemical probe KPU-244-B3 labeled both α- and β-tubulin

Plinabulin (1, NPI-2358), a potent microtubule-targeting agent derived from the natural diketopiperazine ‘phenylahistin’ with a colchicine-like tubulin depolymerization activity, is an anticancer agent undergoing Phase II clinical trials in four countries including the United States. In order to understand the precise binding mode of plinabulin with tubulin, a new bioactive biotin-tagged photoaffinity probe 4 (KPU-244-B3) was designed and synthesized. Probe 4 showed significant binding affinity to tubulin in a binding assay, and selectively bound to tubulin in an HT-1080 cell lysate without photo-irradiation. In a tubulin photoaffinity labeling study, probe 4 labeled both α- and β-tubulin subunits and these interactions were competitively inhibited by plinabulin during photo-irradiation. These results suggest that plinabulin binds in the boundary region between α- and β-tubulin near the colchicine binding site, and not inside the colchicine binding cavity.