A novel O-methyltransferase Cp4MP-OMT catalyses the final step in the biosynthesis of the volatile 1,4-dimethoxybenzene in pumpkin (Cucurbita pepo) flowers.

BACKGROUND: Floral scents play a crucial role in attracting insect pollinators. Among the compounds attractive to pollinators is 1,4-dimethoxybenzene (1,4-DMB). It is a significant contributor to the scent profile of plants from various genera, including economically important Cucurbita species. Despite its importance, the biosynthetic pathway for the formation of 1,4-DMB was not elucidated so far. RESULTS: In this study we showed the catalysis of 1,4-DMB in the presence of 4-methoxyphenol (4-MP) by protein extract from Styrian oil pumpkin (Cucurbita pepo) flowers. Based on this finding, we identified a novel O-methyltransferase gene, Cp4MP-OMT, whose expression is highly upregulated in the volatile-producing tissue of pumpkin flowers when compared to vegetative tissues. OMT activity was verified by purified recombinant Cp4MP-OMT, illustrating its ability to catalyse the methylation of 4-MP to 1,4-DMB in the presence of cofactor SAM (S-(5'-adenosyl)-L-methionine). CONCLUSIONS: Cp4MP-OMT is a novel O-methyltransferase from C. pepo, responsible for the final step in the biosynthesis of the floral scent compound 1,4-DMB. Considering the significance of 1,4-DMB in attracting insects for pollination and in the further course fruit formation, enhanced understanding of its biosynthetic pathways holds great promise for both ecological insights and advancements in plant breeding initiatives.

Mechanism of cytokinesis failure in ovarian cystadenomas with defective BRCA1 and P53 pathways.

We previously described an in vitro model in which serous ovarian cystadenomas were transfected with SV40 large T antigen, resulting in loss of RB and P53 functions and thus mimicking genetic defects present in early high-grade serous extra-uterine Müllerian (traditionally called high-grade serous ovarian) carcinomas including those associated with the BRCA1 mutation carrier state. We showed that replicative aging in this cell culture model leads to a mitotic arrest at the spindle assembly checkpoint. Here we show that this arrest is due to a reduction in microtubule anchoring that coincides with decreased expression of the BUB1 kinase and of the phosphorylated form of its substrate, BUB3. The ensuing prolonged mitotic arrest leads to cohesion fatigue resulting in cell death or, in cells that recover from this arrest, in cytokinesis failure and polyploidy. Down-regulation of BRCA1 to levels similar to those present in BRCA1 mutation carriers leads to increased and uncontrolled microtubule anchoring to the kinetochore resulting in overcoming the spindle assembly checkpoint. Progression to anaphase under those conditions is associated with formation of chromatin bridges between chromosomal plates due to abnormal attachments to the kinetochore, significantly increasing the risk of cytokinesis failure. The dependence of this scenario on accelerated replicative aging can, at least in part, account for the site specificity of the cancers associated with the BRCA1 mutation carrier state, as epithelia of the mammary gland and of the reproductive tract are targets of cell-nonautonomous consequences of this carrier state on cellular proliferation associated with menstrual cycle progressions.

Serum from dengue virus-infected patients with and without plasma leakage differentially affects endothelial cells barrier function in vitro.

BACKGROUND: Although most of cases of dengue infections are asymptomatic or mild symptomatic some individuals present warning signs progressing to severe dengue in which plasma leakage is a hallmark. METHODOLOGY/PRINCIPAL FINDINGS: The present study used Electric Cell-substrate Impedance Sensing (ECIS®) which allows for electrical monitoring of cellular barrier function measuring changes in Transendothelial Electric Resistance (TEER) to investigate the parameters associated with dengue induced leakage. Three groups of individuals were tested: dengue-positives with plasma leakage (leakage), dengue-positives without plasma leakage (no leakage), and dengue-negatives (control). Data show that TEER values of human umbilical vein endothelial cells (HUVECs) was significantly lower after incubation with serum from subjects of the leakage group in comparison to the no leakage or control groups. The serum levels of CXCL1, EGF, eotaxin, IFN-γ, sCD40L, and platelets were significantly decreased in the leakage group, while IL-10, IL-6, and IP-10 levels were significantly increased. We also found a strong correlation between TEER values and augmented levels of IP-10, GM-CSF, IL-1α, and IL-8, as well as decreased levels of CXCL1 and platelets. CONCLUSIONS/SIGNIFICANCE: The present work shows that the magnitude of the immune response contributes to the adverse plasma leakage outcomes in patients and that serum components are important mediators of changes in endothelial homeostasis during dengue infections. In particular, the increased levels of IP-10 and the decreased levels of CXCL1 and platelets seem to play a significant role in the disruption of vascular endothelium associated with leakage outcomes after DENV infection. These findings may have important implications for both diagnostic and therapeutic approaches to predict and mitigate vascular permeabilization in those experiencing the most severe clinical disease outcomes after dengue infection.

The effect of translationally controlled tumour protein (TCTP) on programmed cell death in plants.

BACKGROUND: Translationally controlled tumour protein (TCTP), a well known protein of the animal kingdom, was shown to be a Ca(2+)-binding protein with important functions in many different cellular processes (e.g. protection against stress and apoptosis, cell growth, cell cycle progression, and microtubule organization). However, only little is known about TCTP in plants. Transcript and protein levels of plant TCTPs were shown to be altered by various stress conditions (e.g. cold, salt, draught, aluminium, and pathogen infection), and Arabidopsis thaliana TCTP (AtTCTP) was described as an important regulator of growth. The aim of this study was to further characterize plant TCTP relating to one of its major functions in animals: the protection against cell death. RESULTS: We used two different activators of programmed cell death (PCD) in plants: the mammalian pro-apoptotic protein BAX and tunicamycin, an inhibitor of glycosylation and trigger of unfolded protein response (UPR). Over-expression of AtTCTP significantly decreased cell death in tobacco leaf discs in both studies. A (45)Ca overlay assay showed AtTCTP to be a Ca(2+)-binding protein and localization experiments revealed cytosolic distribution of AtTCTP-GFP in Arabidopsis seedlings. CONCLUSIONS: Our study showed cytoprotective effects of plant TCTP for the first time. Furthermore, we showed the ability of AtTCTP to bind to Ca(2+) and its cytosolic distribution within the cell. If these results are combined, two putative modes of action can be assumed: 1) AtTCTP acts as Ca(2+) sequester, preventing PCD by reducing cytosolic Ca(2+) levels as described for animals. 2) AtTCTP could directly or indirectly interact with other cytosolic or membrane-bound proteins of the cell death machinery, thereby inhibiting cell death progression. As no homologous proteins of the anti-apoptotic machinery of animals were found in plants, and functional homologues still remain to be elucidated, future work will provide more insight.

Role of BRCA1 in controlling mitotic arrest in ovarian cystadenoma cells.

Cancers that develop in BRCA1 mutation carriers are usually near tetraploid/polyploid. This led us to hypothesize that BRCA1 controls the mitotic checkpoint complex, as loss of such control could lead to mitotic errors resulting in tetraploidy/polyploidy and subsequent aneuploidy. We used an in vitro system mimicking premalignant conditions, consisting of cell strains derived from the benign counterparts of serous ovarian carcinomas (cystadenomas) and expressing SV40 large T antigen, conferring the equivalent of a p53 mutation. We previously showed that such cells undergo one or several doublings of their DNA content, as they age in culture and approach the phenomenon of in vitro crisis. Here, we show that such increase in DNA content reflects a cell cycle arrest possibly at the anaphase promoting complex, as evidenced by decreased BrdU incorporation and increased expression of the mitotic checkpoint complex. Down-regulation of BRCA1 in cells undergoing crisis leads to activation of the anaphase promoting complex and resumption of growth kinetics similar to those seen in cells before they reach crisis. Cells recovering from crisis after BRCA1 down-regulation become multinucleated, suggesting that reduced BRCA1 expression may lead to initiation of a new cell cycle without completion of cytokinesis. This is the first demonstration that BRCA1 controls a physiological arrest at the M phase apart from its established role in DNA damage response, a role that could represent an important mechanism for acquisition of aneuploidy during tumor development. This may be particularly relevant to cancers that have a near tetraploid/polyploid number of chromosomes.

Investigations on N-rich protein (NRP) of Arabidopsis thaliana under different stress conditions.

Stress response and adaptation are important physiological mechanisms in plants. As plants are not able to avoid stressful environments by moving away, as animals, they have developed diverse mechanisms to respond to stressful situations. One of the genes involved in these mechanisms is NRP (Asparagine-rich protein or N-rich protein). In this study, NRP expression, protein localization and nrp knockout plants were investigated for further understanding of NRP function. NaCl-induced salt stress, oxidative stress (ozone exposure) and mechanical perturbation (touch treatment) were used to induce abiotic stress. NRP expression was up-regulated in the early phase of stress response to all three elicitors. Stressed nrp knockout seedlings revealed a more pronounced growth inhibition compared to wildtype (salt and osmotic stress). Seedlings showed NRP-GFP expression in the apical meristem, leaf veins, central cylinder, root hair zone and root tip. Analyses of NRP-GFP localization in root cells and protoplasts revealed cytosolic distribution under non-stress conditions and translocation of NRP-GFP to mitochondria due to stress response. Summarizing, our findings point to a contribution of NRP in signal transduction of the initial phase of general stress response in Arabidopsis thaliana.

Cloning of Glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars.

Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a K(m) of 0.7 mm. It requires ATP as phosphate donor (K(m) 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum.

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min(-1). Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.

Effect of pH on the rate of myosin head detachment in molluscan catch muscle: are myosin heads involved in the catch state?

Moderate alkalisation is known to terminate the catch state of bivalve mollusc smooth muscles such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis L. In the present study, we investigated the effect of moderate alkalisation (pH 7.2-7.7 vs control pH 6.7) on the myosin head detachment rate in saponin-skinned fibre bundles of ABRM in order to investigate the possible role of myosin heads in the force maintenance during catch. The detachment rate of myosin heads was deduced from two types of experiments. (1) In stretch experiments on maximally Ca2+-activated fibre bundles (pCa 4.5), the rate of force decay after stepwise stretch was assessed. (2) In ATP step experiments, the rate of force decay from high force rigor (pCa>8) was evaluated. The ATP step was induced by photolysis of caged ATP. We found that moderate alkalisation induces relaxation of skinned fibres in catch, thereby reducing both force and stiffness, whereas it does not accelerate the rate of myosin head detachment. This acceleration, however, would be expected if catch would be simply due to myosin heads remaining sustainably attached to actin filaments. Thus, the myosin heads may be less involved in catch than generally assumed. Catch may possibly depend on a different kind of myofilament interconnections, which are abolished by moderate alkalisation.

No effect of twitchin phosphorylation on the rate of myosin head detachment in molluscan catch muscle: are myosin heads involved in the catch state?

Phosphorylation of twitchin is known to abolish the catch state of anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis. To investigate the role of myosin head involvement in force maintenance during catch, the effect of twitchin phosphorylation on myosin head detachment was studied in saponin-skinned fibre bundles of ABRM. The detachment rate of myosin heads was deduced from two types of experiments: (1) force decay after stepwise stretch of maximally Ca2+-activated fibre bundles (pCa 4.5) and (2) force decay from high-force rigor, the former induced by a stepwise increase in ATP concentration elicited by photolysis of caged ATP (pCa<8). The rate of detachment was not affected by thiophosphorylation or phosphorylation of twitchin by 0.12 mM cAMP in the presence of the phosphatase inhibitor cyclosporine A (1 microM). Conversely, measurements of the rate of stretch-induced delayed force increase (stretch activation) and of the force increase following an ATP step in low-force rigor (pCa 4.5) suggest that the rate of myosin head attachment decreases after twitchin phosphorylation. We conclude that catch is not due to myosin heads remaining attached to actin filaments, but depends on myofilament interconnections that break down when twitchin is phosphorylated.

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle.

The effects of orthovanadate (V(i)), inorganic phosphate (P(i)) and 2,3-butanedione monoxime (BDM) on tension, force transients and the catch state (passive tension maintenance) were investigated in saponin-skinned fibre bundles of the anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis at pH 6.7. During maximal Ca(2+) activation isometric force was depressed by V(i) (0.03-10 mM), P(i) (10 mM) and BDM (50 mM). Force transients following quick stretches (0.1-0.3% of fibre length) were accelerated substantially by 1 mM V(i), 10 mM P(i) or 50 mM BDM. These compounds also accelerated force responses in experiments in which ATP was released rapidly from caged ATP by flash photolysis at both pCa 4.7 (force rise) and at pCa>8 (force decline). The effects on the catch state were investigated in two types of experiments: (1) Ca(2+) removal after maximal Ca(2+) activation and (2) rapid ATP release during high-force rigor at pCa>8. In both cases rapid relaxation was followed by slow relaxation (slower than 2% of initial force per min). This later slow relaxation (catch) was insensitive to V(i) (1-10 mM), P(i) (10 mM) and BDM (50 mM) but was accelerated by 0.12 mM cAMP. Complete relaxation to almost zero force was attained by changing pH from 6.7 to 7.7 (pCa>8). We conclude that catch depends on cAMP- and pH-sensitive structures linking the myofilaments and not on the force-generating actomyosin cross-bridges that are sensitive to V(i), P(i) and BDM.

Improved biochemical disease-free survival of men younger than 60 years with prostate cancer treated with high dose conformal external beam radiotherapy.

PURPOSE: We report the long-term prostate specific antigen relapse-free survival rates and predictors of biochemical outcome for patients 60 years or younger with prostate cancer treated with high dose conformal external beam radiotherapy. MATERIALS AND METHODS: We retrospectively reviewed the records of 740 patients with prostate cancer treated with 3-dimensional conformal radiotherapy or intensity modulated external beam radiotherapy. Patients who also received androgen deprivation therapy were excluded from this analysis. Median radiation dose was 75.6 Gy and median followup was 88 months with a minimum followup of 24 months. Median followup for patients 60 years or younger in this report was 54 months (range 24 to 132). Biochemical failure was defined according to the criteria recommended by the American Society for Therapeutic Radiology and Oncology Consensus Panel. RESULTS: Biochemical failure developed in 20 (21%) of the 96 men 60 years or younger, which was similar to the 22% failure rate observed in 644 patients older than 60. The 5 and 7-year biochemical disease-free survival rates were 82% and 79% in younger men, and 79% and 78% in older men, respectively (p = 0.48). For younger patients who received 81 Gy or greater, the 7-year prostate specific antigen relapse-free survival rates for favorable, intermediate and unfavorable risk patients were 96%, 87% and 50%, respectively. Multivariate analysis revealed that among patients 60 years or younger the most important predictor of biochemical relapse was radiation doses less than 75.6 Gy followed by Gleason score greater than 7. CONCLUSIONS: Men with prostate cancer 60 years or younger treated with high dose radiotherapy have an excellent biochemical outcome and fare as well as older patients. The use of conventional dose levels in patients 60 years or younger was associated with an 8-fold increase in the biochemical relapse rate and these doses should not be considered appropriate for the treatment of localized prostate cancer.

Improved conformality and decreased toxicity with intraoperative computer-optimized transperineal ultrasound-guided prostate brachytherapy.

PURPOSE: We have developed an intraoperative three-dimensional (3D) conformal treatment planning system for permanent prostate implantation in an effort to reduce toxicity further and improve the accuracy of this procedure. We report the preliminary outcome of patients with localized prostate cancer treated with this approach. METHODS AND MATERIALS: Two hundred forty-eight patients with clinically localized prostate cancer were treated with transperineal ultrasound-guided permanent prostate implantation using a real-time intraoperative 3D conformal technique (I-3D) between 1997 and 2001. A genetic algorithm optimization program intraoperatively evaluated the dose deposited throughout the entire 3D volume for multiple seed configurations to identify which seed-loading pattern adhered best to the predetermined target, urethral and rectal dose constraints. The median follow-up time in these patients was 27 months (range 12-51). The dosimetric outcome and acute toxicity profile of these 248 patients were compared with those of patients who were treated between 1988 and 1996 at our institution with a preplanned transperineal implantation technique (PP). RESULTS: Postimplantation dosimetric analysis of the I-3D group demonstrated that the median value of the percentage of the target volume treated to at least the prescription dose (V(100)) was 96%, and the target coverage with the prescription dose (PD) was

High-dose intensity modulated radiation therapy for prostate cancer: early toxicity and biochemical outcome in 772 patients.

PURPOSE: To report the acute and late toxicity and preliminary biochemical outcomes in 772 patients with clinically localized prostate cancer treated with high-dose intensity-modulated radiotherapy (IMRT). METHODS AND MATERIALS: Between April 1996 and January 2001, 772 patients with clinically localized prostate cancer were treated with IMRT. Treatment was planned using an inverse-planning approach, and the desired beam intensity profiles were delivered by dynamic multileaf collimation. A total of 698 patients (90%) were treated to 81.0 Gy, and 74 patients (10%) were treated to 86.4 Gy. Acute and late toxicities were scored by the Radiation Therapy Oncology Group morbidity grading scales. PSA relapse was defined according to The American Society of Therapeutic Radiation Oncology Consensus Statement. The median follow-up time was 24 months (range: 6-60 months). RESULTS: Thirty-five patients (4.5%) developed acute Grade 2 rectal toxicity, and no patient experienced acute Grade 3 or higher rectal symptoms. Two hundred seventeen patients (28%) developed acute Grade 2 urinary symptoms, and one experienced urinary retention (Grade 3). Eleven patients (1.5%) developed late Grade 2 rectal bleeding. Four patients (0.1%) experienced Grade 3 rectal toxicity requiring either one or more transfusions or a laser cauterization procedure. No Grade 4 rectal complications have been observed. The 3-year actuarial likelihood of >/= late Grade 2 rectal toxicity was 4%. Seventy-two patients (9%) experienced late Grade 2 urinary toxicity, and five (0.5%) developed Grade 3 urinary toxicity (urethral stricture). The 3-year actuarial likelihood of >/= late Grade 2 urinary toxicity was 15%. The 3-year actuarial PSA relapse-free survival rates for favorable, intermediate, and unfavorable risk group patients were 92%, 86%, and 81%, respectively. CONCLUSIONS: These data demonstrate the feasibility of high-dose IMRT in a large number of patients. Acute and late rectal toxicities seem to be significantly reduced compared with what has been observed with conventional three-dimensional conformal radiotherapy techniques. Short-term PSA control rates seem to be at least comparable to those achieved with three-dimensional conformal radiotherapy at similar dose levels. Based on this favorable risk:benefit ratio, IMRT has become the standard mode of conformal treatment delivery for localized prostate cancer at our institution.