The DROL1 subunit of U5 snRNP in the spliceosome is specifically required to splice AT-AC-type introns in Arabidopsis.

An Arabidopsis mutant named defective repression of OLE3::LUC 1 (drol1) was originally isolated as a mutant with defects in the repression of OLEOSIN3 (OLE3) after seed germination. In this study, we show that DROL1 is an Arabidopsis homolog of yeast DIB1, a subunit of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome. It is also part of a new subfamily that is specific to a certain class of eukaryotes. Comprehensive analysis of the intron splicing using RNA sequencing analysis of the drol1 mutants revealed that most of the minor introns with AT-AC dinucleotide termini had reduced levels of splicing. Only two nucleotide substitutions from AT-AC to GT-AG enabled AT-AC-type introns to be spliced in drol1 mutants. Forty-eight genes, including those having important roles in abiotic stress responses and cell proliferation, exhibited reduced splicing of AT-AC-type introns in the drol1 mutants. Additionally, drol1 mutant seedlings showed retarded growth, similar to that caused by the activation of abscisic acid signaling, possibly as a result of reduced AT-AC-type intron splicing in the endosomal Na+ /H+ antiporters and plant-specific histone deacetylases. These results indicate that DROL1 is specifically involved in the splicing of minor introns with AT-AC termini and that this plays an important role in plant growth and development.

Anti-inflammatory Quinoline Alkaloids from the Roots of Waltheria indica.

Sixteen new quinoline alkaloids (1a-7, 8a, 9, 10, 13-15, 17, and 21) and 10 known analogs (8b, 11, 12, 16, 18-20, and 22-24), along with three known cyclopeptide alkaloids (25-27), were isolated from the roots of Waltheria indica. The structures of the new compounds were elucidated by detailed NMR and circular dichroism with computational support and mass spectrometry data interpretation. Anti-inflammatory potential of isolates was evaluated based on inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production and tumor necrosis factor-alpha (TNF-α)-induced nuclear factor kappa B (NF-κB) activity with cell culture models. In the absence of cell growth inhibition, compounds 6, 8a, 9-11, 13, 21, and 24 reduced TNF-α-induced NF-κB activity with IC50 values ranging from 7.1 to 12.1 µM, comparable to the positive control (BAY 11-7082, IC50 = 9.7 µM). Compounds 6, 8a, 8b, and 11 showed significant NO-inhibitory activity with IC50 values ranging from 11.0 to 12.8 µM, being more active than the positive control (l-NMMA, IC50 = 22.7 µM). Structure-activity relationships indicated that NO inhibitory activity was significantly affected by C-8 substitution. Inhibition of LPS-induced nitric oxide synthase (iNOS) by 8b [(5S)-waltherione M, IC50 11.7 ± 0.8 µM] correlated with inhibition of iNOS mRNA expression. The biological potential of W. indica metabolites supports the traditional use of this plant for the treatment of inflammatory-related disorders.

Cine magnetic resonance imaging provides novel predictors of early continence recovery after radical prostatectomy: Assessment of the dynamics of pelvic floor muscles.

AIMS: Postprostatectomy incontinence is a major complication of prostatectomy. Although pelvic floor muscle training can successfully treat postprostatectomy incontinence, evidence for how muscle movement affects continence recovery is lacking. We evaluated dynamic factors of prostatectomy patients using cine magnetic resonance imaging to identify risk factors for postprostatectomy incontinence and reveal the contribution of pelvic floor muscles to continence recovery. METHODS: A total of 128 prostate cancer patients who underwent robot-assisted laparoscopic surgery were enrolled. Cine magnetic resonance imaging was performed preoperatively and 6 months after surgery. Continence was defined as pad-free or use of safety pads. We defined the bladder neck elevation distance during pelvic floor muscle training as the bladder elevation distance. Patients with continence recovery within 1 month comprised the continence group (n = 48); other patients comprised the incontinence group (n = 80). RESULTS: The preoperative bladder elevation distance was significantly longer in the continence group than in the incontinence group (10.4 vs. 8.2 mm; p < .001). The postoperative bladder elevation distance of the continence group tended to be longer (9.9 vs. 8.9 mm; p = .057). Multivariate analysis showed that the preoperative bladder elevation distance significantly contributed to continence recovery (p = .016). Patients with a longer preoperative bladder elevation distance (>8.5 mm) experienced continence recovery significantly faster than patients with a shorter distance (<8.5mm) (p = .038). CONCLUSIONS: Bladder elevation distance, a novel dynamic parameter, was strongly associated with early continence recovery. Cine magnetic resonance imaging can assess a patient's risk of postprostatectomy incontinence and guide pelvic floor muscle training.

The PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots.

Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis thaliana root, which contrasts with well-described transcription factor gradients in animals specifying distinct cell fates within an essentially static context. To understand the output of the PLT gradient, we studied the gene set transcriptionally controlled by PLTs. Our work reveals how the PLT gradient can regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation. Moreover, PLT targets include major patterning genes and autoregulatory feedback components, enforcing their role as master regulators of organ development.

A duplicated NUCLEOLIN gene with antagonistic activity is required for chromatin organization of silent 45S rDNA in Arabidopsis.

In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.

[MODIFICATIONS FOR CONTROLLING THE DORSAL VEIN COMPLEX IN ROBOTIC-ASSISTED LAPAROSCOPIC RADICAL PROSTATECTOMY].

OBJECTIVE: To investigate a superior method for controlling the dorsal vein complex (DVC) in robotic-assisted laparoscopic radical prostatectomy at Nagakubo hospital. PATIENTS AND METHODS: DVC control was altered from suture ligation followed by DVC division (SL group, n = 115) to preceding division followed by suture (PD group, n = 62). Surgical margin status and functional outcomes were investigated. RESULTS: No notable surgical complications were found in the PD group. Operation time and estimated blood loss were significantly less in the PD group; however, changes to serum hemoglobin before and after surgery showed no significant difference. The rates of positive surgical margin were 26.1% and 11.3% in the SL and PD groups, respectively (p = 0.021). Apical margin positivity was 9.6% and 3.2% in the SL and PD groups, respectively (p = 0.12). Urinary continence outcomes of within one pad/day and pad free in the SL and PD groups until 6 mo after surgery were 93.6% and 89.1% (P = 0.21), and 67.3% and 67.5% (p = 0.96), respectively. Erection recovery within 6 mo was 43.6% and 41.7% in the SL and PD groups (p = 0.91). CONCLUSION: Preceding division followed by suture method decreased apical margin positivity and provided superior cancer control in comparison with suture ligation followed by DVC division, although, it was probably influenced by the learning curve. Functional outcomes were not significantly different with either method.

Wound-induced expression of DEFECTIVE IN ANTHER DEHISCENCE1 and DAD1-like lipase genes is mediated by both CORONATINE INSENSITIVE1-dependent and independent pathways in Arabidopsis thaliana.

Endogenous JA production is not necessary for wound-induced expression of JA-biosynthetic lipase genes such as DAD1 in Arabidopsis. However, the JA-Ile receptor COI1 is often required for their JA-independent induction. Wounding is a serious event in plants that may result from insect feeding and increase the risk of pathogen infection. Wounded plants produce high amounts of jasmonic acid (JA), which triggers the expression of insect and pathogen resistance genes. We focused on the transcriptional regulation of DEFECTIVE IN ANTHER DEHISCENCE1 and six of its homologs including DONGLE (DGL) in Arabidopsis, which encode lipases involved in JA biosynthesis. Plants constitutively expressing DAD1 accumulated a higher amount of JA than control plants after wounding, indicating that the expression of these lipase genes contributes to determining JA levels. We found that the expression of DAD1, DGL, and other DAD1-LIKE LIPASE (DALL) genes is induced upon wounding. Some DALLs were also expressed in unwounded leaves. Further experiments using JA-biosynthetic and JA-response mutants revealed that the wound induction of these genes is regulated by several distinct pathways. DAD1 and most of its homologs other than DALL4 were fully induced without relying on endogenous JA-Ile production and were only partly affected by JA deficiency, indicating that positive feedback by JA is not necessary for induction of these genes. However, DAD1 and DGL required CORONATINE INSENSITIVE1 (COI1) for their expression, suggesting that a molecule other than JA might act as a regulator of COI1. Wound induction of DALL1, DALL2, and DALL3 did not require COI1. This differential regulation of DAD1 and its homologs might explain their functions at different time points after wounding.

[Comparison of initial results with robotic-assisted laparoscopic radical prostatectomy and open retropubic radical prostatectomy].

OBJECTIVE: To investigate the superiority in 2 radical prostatectomies, we compared the initial results of robotic-assisted radical prostatectomy (RARP) to those of retropubic radical prostatectomy (RRP) performed during the same period at Nagakubo hospital. PATIENTS AND METHODS: The study was conducted on a total of 160 patients having undergone radical prostatectomy from April 2009 to March 2012 (92 patients with RARP and 68 with RRP). We investigated surgical stress, cancer control, functional outcomes and complications in both groups. RESULTS: Surgical stress; operation time was significantly shorter with RRP; however, blood loss and serum total protein loss were significantly less with RARP. White blood cell count at 2 days after surgery was significantly less with RARP. The rates of analgesic use and SIRS were similar. Although the date on which taking solid meals resumed did not differ, the duration of indwelling urethral catheter and admission period were significantly shorter with RARP. Cancer control; the rates of positive surgical margin were 27.2% and 19.1% with RARP and RRP, respectively (p = 0.24), and biochemical recurrence was seen in 12.0% and 19.1% with RARP and RRP, respectively (p = 0.73), which were not significantly different. Continence; urinary continence outcomes with RARP and RRP were 17% and 4% for urinary continence at discharge (p = 0.01), 1.8 and 3.3 months for no more than one pad per day (p < 0.01), and 4.3 and 6.2 months for pad free (p = 0.03), respectively. Sexual function; erection recovery within 6 mo was only observed with RARP; however, overall recovery rate of erection was 65% and 75% with RARP and RRP, respectively (p = 0.69). COMPLICATIONS: 1 case with a rectal injury was seen in both groups, but complication rates were 8.7% and 16.2% with RARP and RRP, respectively (p = 0.22). CONCLUSION: In spite of our initial experience of RARP, surgical stress and complications with RARP were considered to be superior to that with RRP. Cancer control and sexual function showed no significant difference between RARP and RRP, however, urinary continence outcome is significantly superior with RARP. Our data suggest that treatment outcome after initial experience with RARP is not inferior to that with RRP, and better results are expected by improving surgical techniques.

AtNUFIP, an essential protein for plant development, reveals the impact of snoRNA gene organisation on the assembly of snoRNPs and rRNA methylation in Arabidopsis thaliana.

In all eukaryotes, C/D small nucleolar ribonucleoproteins (C/D snoRNPs) are essential for methylation and processing of ribosomal RNAs. They consist of a box C/D small nucleolar RNA (C/D snoRNA) associated with four highly conserved nucleolar proteins. Recent data in HeLa cells and yeast have revealed that assembly of these snoRNPs is directed by NUFIP protein and other auxiliary factors. Nevertheless, the precise function and biological importance of NUFIP and the other assembly factors remains unknown. In plants, few studies have focused on RNA methylation and snoRNP biogenesis. Here, we identify and characterise the AtNUFIP gene that directs assembly of C/D snoRNP. To elucidate the function of AtNUFIP in planta, we characterized atnufip mutants. These mutants are viable but have severe developmental phenotypes. Northern blot analysis of snoRNA accumulation in atnufip mutants revealed a specific degradation of C/D snoRNAs and this situation is correlated with a reduction in rRNA methylation. Remarkably, the impact of AtNUFIP depends on the structure of snoRNA genes: it is essential for the accumulation of those C/D snoRNAs encoded by polycistronic genes, but not by monocistronic or tsnoRNA genes. We propose that AtNUFIP controls the kinetics of C/D snoRNP assembly on nascent precursors to overcome snoRNA degradation of aberrant RNPs. Finally, we show that AtNUFIP has broader RNP targets, controlling the accumulation of scaRNAs that direct methylation of spliceosomal snRNA in Cajal bodies.

Mutations in MYB3R1 and MYB3R4 cause pleiotropic developmental defects and preferential down-regulation of multiple G2/M-specific genes in Arabidopsis.

R1R2R3-Myb proteins represent an evolutionarily conserved class of Myb family proteins important for cell cycle regulation and differentiation in eukaryotic cells. In plants, this class of Myb proteins are believed to regulate the transcription of G2/M phase-specific genes by binding to common cis-elements, called mitosis-specific activator (MSA) elements. In Arabidopsis (Arabidopsis thaliana), MYB3R1 and MYB3R4 act as transcriptional activators and positively regulate cytokinesis by activating the transcription of KNOLLE, which encodes a cytokinesis-specific syntaxin. Here, we show that the double mutation myb3r1 myb3r4 causes pleiotropic developmental defects, some of which are due to deficiency of KNOLLE whereas other are not, suggesting that multiple target genes are involved. Consistently, microarray analysis of the double mutant revealed altered expression of many genes, among which G2/M-specific genes showed significant overrepresentation of the MSA motif and a strong tendency to be down-regulated by the double mutation. Our results demonstrate, on a genome-wide level, the importance of the MYB3R-MSA pathway for regulating G2/M-specific transcription. In addition, MYB3R1 and MYB3R4 may have diverse roles during plant development by regulating G2/M-specific genes with various functions as well as genes possibly unrelated to the cell cycle.

Photoselective vaporization of the prostate: pursuing good indications based on the results of 400 Japanese patients.

OBJECTIVE: To investigate good indications of photoselective vaporization of the prostate (PVP) by retrospective evaluation of its efficacy in relation to prostate volume. PATIENTS AND METHODS: A cohort of 400 patients had International Prostate Symptoms Score (IPSS), Quality of Life (QoL) score, urinary peak flow (Q(max) ), and prostate volume (P(vol) ), assessed before and after surgery. Changes of outcome variables were compared among four groups classified according to preoperative prostate volume: <30 mL (group A), ≥30 and <50 mL (group B), ≥50 and <70 mL (group C), and >70 mL (group D). RESULTS: Operating times and amounts of energy applied were 44.2 min and 159 kJ, 67.7 min and 268 kJ, 111 min and 409 kJ, and 171 min and 604 kJ in groups A, B, C, and D, respectively. All variables improved significantly and immediately, and remained for more than 12 months. Investigation of groups classified by prostate size demonstrated that IPSS and QoL score decreased and improvement rate of Q(max) rose as prostate volume increased. Changes of P(vol) were from 24.5 mL to 13.2 mL (-46.1%), from 39.8 mL to 22.9 mL (-42.4%), from 58.4 mL to 32.8 mL (-43.8%), and from 92.1 mL to 53.8 mL (-41.5%) in groups A, B, C, and D, respectively. Overall the P(vol) reduction rate was 43.2%, and no significant differences were observed in reduction ratios among the groups. Complications were minimal, but bladder neck contracture arose in 16 cases with a smaller prostate. CONCLUSION: Whilst the percentage reduction in volume was similar across the groups, the larger absolute volume reduction in patients with larger prostates was associated with greater improvement in objective urinary function variables, and with symptom score reduction.

Transgenic sweet potato expressing thionin from barley gives resistance to black rot disease caused by Ceratocystis fimbriata in leaves and storage roots.

Black rot of sweet potato caused by pathogenic fungus Ceratocystis fimbriata severely deteriorates both growth of plants and post-harvest storage. Antimicrobial peptides from various organisms have broad range activities of killing bacteria, mycobacteria, and fungi. Plant thionin peptide exhibited anti-fungal activity against C. fimbriata. A gene for barley α-hordothionin (αHT) was placed downstream of a strong constitutive promoter of E12Ω or the promoter of a sweet potato gene for ß-amylase of storage roots, and introduced into sweet potato commercial cultivar Kokei No. 14. Transgenic E12Ω:αHT plants showed high-level expression of αHT mRNA in both leaves and storage roots. Transgenic ß-Amy:αHT plants showed sucrose-inducible expression of αHT mRNA in leaves, in addition to expression in storage roots. Leaves of E12Ω:αHT plants exhibited reduced yellowing upon infection by C. fimbriata compared to leaves of non-transgenic Kokei No. 14, although the level of resistance was weaker than resistance cultivar Tamayutaka. Storage roots of both E12Ω:αHT and ß-Amy:αHT plants exhibited reduced lesion areas around the site inoculated with C. fimbriata spores compared to Kokei No. 14, and some of the transgenic lines showed resistance level similar to Tamayutaka. Growth of plants and production of storage roots of these transgenic plants were not significantly different from non-transgenic plants. These results highlight the usefulness of transgenic sweet potato expressing antimicrobial peptide to reduce damages of sweet potato from the black rot disease and to reduce the use of agricultural chemicals.

A link among DNA replication, recombination, and gene expression revealed by genetic and genomic analysis of TEBICHI gene of Arabidopsis thaliana.

Spatio-temporal regulation of gene expression during development depends on many factors. Mutations in Arabidopsis thaliana TEBICHI (TEB) gene encoding putative helicase and DNA polymerase domains-containing protein result in defects in meristem maintenance and correct organ formation, as well as constitutive DNA damage response and a defect in cell cycle progression; but the molecular link between these phenotypes of teb mutants is unknown. Here, we show that mutations in the DNA replication checkpoint pathway gene, ATR, but not in ATM gene, enhance developmental phenotypes of teb mutants, although atr suppresses cell cycle defect of teb mutants. Developmental phenotypes of teb mutants are also enhanced by mutations in RAD51D and XRCC2 gene, which are involved in homologous recombination. teb and teb atr double mutants exhibit defects in adaxial-abaxial polarity of leaves, which is caused in part by the upregulation of ETTIN (ETT)/AUXIN RESPONSIVE FACTOR 3 (ARF3) and ARF4 genes. The Helitron transposon in the upstream of ETT/ARF3 gene is likely to be involved in the upregulation of ETT/ARF3 in teb. Microarray analysis indicated that teb and teb atr causes preferential upregulation of genes nearby the Helitron transposons. Furthermore, interestingly, duplicated genes, especially tandemly arrayed homologous genes, are highly upregulated in teb or teb atr. We conclude that TEB is required for normal progression of DNA replication and for correct expression of genes during development. Interplay between these two functions and possible mechanism leading to altered expression of specific genes will be discussed.

[Hemodialysis case in which RCC was identified after disappearance of perirenal hematoma following spontaneous renal rupture treated by transcatheter arterial embolization].

A 55-year-old man who had been undergoing hemodialysis for 9 years visited our institution after the sudden onset of severe left flank pain. He presented with hypotension and was admitted immediately because computed tomography (CT) revealed a massive perirenal hematoma. Renal arteriography showed contrast media leakage at the lower branch of the left renal artery, and spontaneous renal rupture was diagnosed. Five months after the bleeding was stopped by selective transcatheter embolization of the branch of renal artery, CT showed an enhanced mass at the upper pole of left kidney and renal cell carcinoma (RCC) was suspected. Radical nephrectomy was performed, the pathological diagnosis was clear cell carcinoma, and the man has not experienced recurrence within 36 months after the surgery. RCC did not appear to be the cause of the original hemorrhage because there was a small residual hematoma in the middle of the renal parenchyma that was separated from the RCC. In cases of spontaneous renal rupture, re-evaluation by imaging studies is mandatory after disappearance of perirenal hematoma because imaging studies at the time of the rupture sometimes do not reveal the cause of the hemorrhage.

An AP2-type transcription factor, WRINKLED1, of Arabidopsis thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis.

Although an APETALA2 (AP2)-type transcription factor, WRINKLED1 (WRI1), has been shown to be required for accumulation of triacylglycerols (TAGs) in Arabidopsis seeds, its direct target genes have not been established. Overexpression of WRI1 up-regulated a set of genes involved in fatty acid (FA) synthesis in plastids, including genes for a subunit of pyruvate kinase (Pl-PKbeta1), acetyl-CoA carboxylase (BCCP2), acyl carrier protein (ACP1), and ketoacyl-acyl carrier protein synthase (KAS1), while expression of these genes is reduced in mutants with reduced WRI1 expression. Transient expression of LUC reporter genes with the proximal sequences upstream from the ATG codon of Pl-PKbeta1, BCCP2, and KAS1 in protoplasts was activated by co-expression of WRI1, and recombinant WRI1 bound to these upstream sequences in vitro. The seven WRI1 binding sites shared a sequence [CnTnG](n)(7)[CG], where n is any nucleotide designated as the AW-box, and mutations in AW-boxes near the transcription start site and in the 5'-untranslated region of Pl-PKbeta1 abolished activation by WRI1 in protoplasts and expression during seed maturation. Although expression of genes for the synthesis of TAGs and packaging into oil bodies in the endoplasmic reticulum in developing seeds required WRI1, their expression was not up-regulated by WRI1 overexpression. Thus, WRI1 promotes the flow of carbon to oil during seed maturation by directly activating genes involved in FA synthesis and controlling genes for assembly and storage of TAG.

The Arabidopsis FLAKY POLLEN1 gene encodes a 3-hydroxy-3-methylglutaryl-coenzyme A synthase required for development of tapetum-specific organelles and fertility of pollen grains.

The pollen coat is a surface component of pollen grains required for fertilization. To study how the pollen coat is produced, we identified and characterized a recessive and conditional male-sterile Arabidopsis mutant, flaky pollen1-1 (fkp1-1), whose pollen grains lack functional pollen coats. FKP1 is a single-copy gene in the Arabidopsis genome and encodes 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMG-CoA synthase), an enzyme of the mevalonate (MVA) pathway involved in biosynthesis of isoprenoids such as sterols. We found that fkp1-1 possesses a T-DNA insertion 550 bp upstream of the initiation codon. RT-PCR and promoter analyses revealed that fkp1-1 results in knockdown of FKP1 predominantly in tapetum. Electron microscopy showed that the mutation affected the development of tapetum-specific lipid-containing organelles (elaioplast and tapetosome), causing the deficient formation of fkp1-1 pollen coats. These results suggest that both elaioplasts, which accumulate vast amount of sterol esters, and tapetosomes, which are unique oil-accumulating structures, require the MVA pathway for development. Null alleles of fkp1 were male-gametophyte lethal upon pollen tube elongation, whereas female gametophytes were normal. These results show that the MVA pathway is essential, at least in tapetal cells and pollen grains, for the development of tapetum-specific organelles and the fertility of pollen grains.

Arabidopsis auxin response factor6 and 8 regulate jasmonic acid biosynthesis and floral organ development via repression of class 1 KNOX genes.

Two mutations in Arabidopsis thaliana, auxin response factor6 (arf6) and arf8, concomitantly delayed the elongation of floral organs and subsequently delayed the opening of flower buds. This phenotype is shared with the jasmonic acid (JA)-deficient mutant dad1, and, indeed, the JA level of arf6 arf8 flower buds was decreased. Among JA biosynthetic genes, the expression level of DAD1 (DEFECTIVE IN ANTHER DEHISCENCE1) was markedly decreased in the double mutant, suggesting that ARF6 and ARF8 are required for activation of DAD1 expression. The double mutant arf6 arf8 also showed other developmental defects in flowers, such as aberrant vascular patterning and lack of epidermal cell differentiation in petals. We found that class 1 KNOX genes were expressed ectopically in the developing floral organs of arf6 arf8, and mutations in any of the class 1 KNOX genes (knat2, knat6, bp and hemizygous stm) partially suppressed the defects in the double mutant. Furthermore, ectopic expression of the STM gene caused a phenotype similar to that of arf6 arf8, including the down-regulation of DAD1 expression. These results suggested that most defects in arf6 arf8 are attributable to abnormal expression of class 1 KNOX genes. The expression of AS1 and AS2 was not affected in arf6 arf8 flowers, and as1 and arf6 arf8 additively increased the expression of class 1 KNOX genes. We concluded that ARF6 and ARF8, in parallel with AS1 and AS2, repress the class 1 KNOX genes in developing floral organs to allow progression of the development of these organs.

[EVALUATING THE RELATIONSHIP BETWEEN LOWER URINARY TRACT SYMPTOMS AND ENDOTHELIAL FUNCTION USING FLOW-MEDIATED DILATION, AND THE EFFECTS OF TADALAFIL].

(Objective) Recently, lower urinary tract symptoms (LUTS) were reported to be associated with endothelial dysfunction. Endothelial function is non-invasively measured by flow-mediated dilation (FMD). As tadalafil has the potential to improve atherosclerosis, we evaluated the relationship between LUTS and endothelial function using FMD, and the effects of tadalafil. (Patients and methods) We conducted FMD examinations for a total of 122 males, and analyzed its association with IPSS, OABSS, and cardiovascular risks. Furthermore, 21 BPH patients received 5 mg of tadalafil per day for one year. We defined the Low FMD group as FMD < 3.9% and the Control group as other values, and compared the effects of tadalafil between groups. (Results) In the 122 male patients, FMD was negatively correlated with nocturia and OABSS. Patients with hypertension or coronary artery disease had a lower FMD than those without.In the tadalafil administration study, the Low FMD group achieved greater improvement of IPSS, OABSS and FMD than the Control group. (Conclusion) FMD examination revealed that endothelial dysfunction is closely associated with LUTS in males, and that tadalafil is effective for patients with endothelial dysfunction.

Identification of kaonashi mutants showing abnormal pollen exine structure in Arabidopsis thaliana.

Exine, the outermost architecture of pollen walls, protects male gametes from the environment by virtue of its chemical and physical stability. Although much effort has been devoted to revealing the mechanism of exine construction, still little is known about it. To identify the genes involved in exine formation, we screened for Arabidopsis mutants with pollen grains exhibiting abnormal exine structure using scanning electron microscopy. We isolated 12 mutants, kaonashi1 (kns1) to kns12, and classified them into four types. The type 1 mutants showed a collapsed exine structure resembling a mutant of the callose synthase gene, suggesting that the type 1 genes are involved in callose wall synthesis. The type 2 mutant showed remarkably thin exine structure, presumably due to defective primexine thickening. The type 3 mutants showed defective tectum formation, and thus type 3 genes are required for primordial tectum formation or biosynthesis and deposition of sporopollenin. The type 4 mutants showed densely distributed baculae, suggesting type 4 genes determine the position of probacula formation. All identified kns mutants were recessive, suggesting that these KNS genes are expressed in sporophytic cells. Unlike previously known exine-defective mutants, most of the kns mutants showed normal fertility. Map-based cloning revealed that KNS2, one of the type 4 genes, encodes sucrose phosphate synthase. This enzyme might be required for synthesis of primexine or callose wall, which are both important for probacula positioning. Analysis of kns mutants will provide new knowledge to help understand the mechanism of biosynthesis of exine components and the construction of exine architecture.

Development of R4 gateway binary vectors (R4pGWB) enabling high-throughput promoter swapping for plant research.

We developed a new series of Gateway binary vectors, R4pGWBs, that are plant transformation vectors designed for one-step construction of chimeric genes between any promoter and any cDNA. The structure of R4pGWBs is almost the same as the promoterless type of improved pGWBs (ImpGWBs), except that the attR1 site is replaced with attR4, which enables tripartite recombination of these vectors with promoter- and cDNA-entry clones. While ImpGWBs are suitable for promoter analysis and constitutive expression of cDNAs in higher plants, R4pGWBs have a great advantage in expressing a cDNA under the regulation of desired promoters.