Emission of herbivore elicitor-induced sesquiterpenes is regulated by stomatal aperture in maize (Zea mays) seedlings.

Maize seedlings emit sesquiterpenes during the day in response to insect herbivory. Parasitoids and predators use induced volatile blends to find their hosts or prey. To investigate the diurnal regulation of biosynthesis and emission of induced sesquiterpenes, we applied linolenoyl-L-glutamine (LG) to maize seedlings in the morning or evening using a cut-stem assay and tracked farnesene emission, in planta accumulation, as well as transcript levels of farnesyl pyrophosphate synthase 3 (ZmFPPS3) and terpene synthase10 (ZmTPS10) throughout the following day. Independent of time of day of LG treatment, maximum transcript levels of ZmFPPS3 and ZmTPS10 occurred within 3-4 h after elicitor application. The similarity between the patterns of farnesene emission and in planta accumulation in light-exposed seedlings in both time courses suggested unobstructed emission in the light. After evening induction, farnesene biosynthesis increased dramatically during early morning hours. Contrary to light-exposed seedlings dark-kept seedlings retained the majority of the synthesized farnesene. Two treatments to reduce stomatal aperture, dark exposure at midday, and abscisic acid treatment before daybreak, resulted in significantly reduced amounts of emitted and significantly increased amounts of in planta accumulating farnesene. Our results suggest that stomata not only play an important role in gas exchange for primary metabolism but also for indirect plant defences.

Upregulation of non-ß cell-derived vascular endothelial growth factor A increases small clusters of insulin-producing cells in the pancreas.

Pancreatic ß cell-derived vascular endothelial growth factor A (VEGF-A) contributes to normal ß cell function. We therefore hypothesized that non-ß cell-derived VEGF-A may affect its properties in adult mice.We generated transgenic mice expressing human VEGF-A (hVEGF-A) in a visceral smooth muscle cell (SMC)-dominant manner under the control of the transgelin (Tagln/SM22α) promoter via a tamoxifen-induced Cre/loxP recombination system (SM-CreER(T2)/hVEGF mice).SM-CreER(T2)/hVEGF mice received tamoxifen orally followed by microscopic examination of their pancreas 4 weeks after the hVEGF-A induction. The number of clusters of insulin-producing cells (IPCs) in islets, pancreatic ducts, and individual IPCs were counted.The number of small IPC clusters (100-215 µm(2)) in the pancreas increased significantly in SM-CreER(T2)/hVEGF mice compared with SM-CreER(T2)(Ki) mice (473 out of 1 992 counts vs. 199 out of 976 counts, p<0.05), although total IPC area and the number of pancreatic duct IPCs, in proportion to exocrine area, were similar between the 2 groups. Although most small IPC clusters observed in SM-CreER(T2)/hVEGF mice were not accompanied by α and/or δ cells, some were attached to a single or a few α cells. An STZ-induced diabetic state in SM-CreER(T2)/hVEGF mice was slightly ameliorated, with only one point of significance 12 weeks after STZ administration, compared with SM-CreER(T2)(Ki) mice.Upregulation of non-ß cell-derived VEGF-A may alter the composition of pancreatic IPCs by increasing the number of small IPC clusters. These findings provide new information on the role of non-ß cell-derived VEGF-A to IPC regeneration and insulin production.

Self-propelled motion of a fluid droplet under chemical reaction.

We study self-propelled dynamics of a droplet due to a Marangoni effect and chemical reactions in a binary fluid with a dilute third component of chemical product which affects the interfacial energy of a droplet. The equation for the migration velocity of the center of mass of a droplet is derived in the limit of an infinitesimally thin interface. We found that there is a bifurcation from a motionless state to a propagating state of droplet by changing the strength of the Marangoni effect.

Polarity patterns of stress fibers.

Stress fibers are contractile actomyosin bundles commonly observed in the cytoskeleton of metazoan cells. The spatial profile of the polarity of actin filaments inside contractile actomyosin bundles is either monotonic (graded) or periodic (alternating). In the framework of linear irreversible thermodynamics, we write the constitutive equations for a polar, active, elastic one-dimensional medium. An analysis of the resulting equations for the dynamics of polarity shows that the transition from graded to alternating polarity patterns is a nonequilibrium Lifshitz point. Active contractility is a necessary condition for the emergence of sarcomeric, alternating polarity patterns.

Different pathways in mechanical unfolding/folding cycle of a single semiflexible polymer.

Kinetics of conformational change of a semiflexible polymer under mechanical external field were investigated with Langevin dynamics simulations. It is found that a semiflexible polymer exhibits large hysteresis in mechanical folding/unfolding cycle even with a slow operation, whereas in a flexible polymer, the hysteresis almost disappears at a sufficiently slow operation. This suggests that the essential features of the structural transition of a semiflexible polymer should be interpreted at least on a two-dimensional phase space. The appearance of such large hysteresis is discussed in relation to different pathways in the loading and unloading processes. By using a minimal two-variable model, the hysteresis loop is described in terms of different pathways on the transition between two stable states.

Antinociceptive effect of the novel compound OT-7100 in a diabetic neuropathy model.

We previously reported that OT-7100 (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-alpha]pyrimidine) had antinociceptive potency in various animal models. To further characterize this compound, the present study examined the effects of OT-7100 on mechanical hyperalgesia and motor nerve conduction velocity in streptozotocin-induced diabetic rats. OT-7100 significantly increased the nociceptive threshold in the diabetic rat in a dose-dependent manner. Gabapentin (anticonvulsant agent) and insulin strongly increased the nociceptive threshold but gabapentin increased it above normal levels. An aldose reductase inhibitor slightly increased the nociceptive threshold at a high dose. We also measured glucose levels and motor nerve conduction velocity in OT-7100-treated rats. Insulin decreased glucose levels but OT-7100 had no effect on glucose levels or on motor nerve conduction velocity. These results suggest that OT-7100 alleviates hyperalgesia in a diabetic neuropathy model in a different manner from gabapentin or aldose reductase inhibitor and may be a new treatment for the pain associated with peripheral nerve injury.

Intercalating fluorescence dye YOYO-1 prevents the folding transition in giant duplex DNA.

Recently, it has become clear that with the addition of polyamines, giant DNA molecules of size greater than 10 kbp exhibit all-or-none switching between elongated coil and folded compact states. Here the effects of the intercalating fluorescent labeling dye, YOYO-1, and the minor-groove binding fluorescent labeling dye, DAPI, on the folding transition of single giant T4 DNA (166 kbp) induced by spermidine(3+) were examined, by use of the experimental technique of single molecular chain observation with fluorescence microscopy. It is found that the intercalating dye, YOYO-1, markedly prevents the folding transition, whereas the minor-groove binding dye, DAPI, exhibits negligible effect on the folding transition. This action of YOYO-1 is discussed in relation to the biological effect of intercalators.

Rapid generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in rat brain after decapitation.

Rat brain, frozen in liquid nitrogen immediately after decapitation, contains a substantial amount of 2-arachidonoylglycerol (0.34 nmol/g tissue), an endogenous cannabinoid receptor ligand. The level of 2-arachidonoylglycerol in the brain was rapidly augmented after decapitation, the peak being noted 30 s after decapitation (1.54 nmol/g tissue). Noticeably, there are two phases during the increase in the levels of 2-arachidonoylglycerol: a rapid transient increase and a subsequent gradual sustained increase, suggesting that at least two separate mechanisms are involved in the generation of 2-arachidonoylglycerol in the decapitated brain. Gradual sustained formation was also observed for other monoacylglycerols, (e.g. 2-palmitoylglycerol plus 2-oleoylglycerol and 2-cis-vaccenoylglycerol). Thus, it is important to minimize post-mortem changes to estimate the exact tissue levels of 2-arachidonoylglycerol as well as other monoacylglycerols in the brain.

A novel analgesic compound OT-7100 attenuates nociceptive responses in animal models of inflammatory and neuropathic hyperalgesia: a possible involvement of adenosinergic anti-nociception.

We studied the effects of OT-7100 (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo [1,5-a]pyrimidine), a novel analgesic compound, on the inhibitory action of adenosine on the contraction of guinea pig ileum and investigated the effects of OT-7100 on the nociceptive responses in animal models of inflammatory and peripheral neuropathic hyperalgesia and decreases spinal c-Fos expression. OT-7100 at 0.3 - 3 microM significantly enhanced the inhibitory effect of adenosine on the contraction of guinea pig ileum. The efficacy of OT-7100 (1, 3 or 10 mg/kg, p.o.) on hyperalgesia induced by yeast or substance P and in the Bennett model was significantly suppressed by coadministration of the adenosine A1 antagonist DPCPX (0.01 or 0.1 pmol/animal, i.t.), while OT-7100 without DPCPX significantly increased the nociceptive threshold in each rat model. OT-7100 (3, 10 and 30 mg/kg per day, p.o.) significantly inhibited the mechanical nociceptive threshold in the injured paw in the Chung model. OT-7100 (30 mg/kg, p.o.) significantly decreased the number of Fos-LI neurons in the spinal dorsal horn in the Bennett model. These finding suggest that OT-7100 inhibits hyperalgesia in these animal models possibly by enhancing adenosinergic neurotransmission in the dorsal horn, although we still lack direct evidence for it.

Generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in picrotoxinin-administered rat brain.

The levels of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, and other molecular species of monoacylglycerols in rat brain were examined. In this study, we sacrificed the animals in liquid nitrogen to minimize postmortem changes. We found that rat brain contains 0.23 nmol/g tissue of 2-arachidonoylglycerol, which accounts for 10.5% of the total monoacylglycerol present in this tissue. We next investigated the level of 2-arachidonoylglycerol after in vivo stimulation with picrotoxinin. We found that the level of 2-arachidonoylglycerol was elevated markedly in picrotoxinin-administered rat brain (4- to 6-fold over the control level). Changes in the levels of other molecular species were relatively small or negligible. Several cannabimimetic molecules as well as Delta(9)-tetrahydrocannabinol are known to depress neurotransmission and to exert anticonvulsant activities; endogenous 2-arachidonoylglycerol produced during neural excitation may play a regulatory role in calming the enhanced synaptic transmission.

Apoptosis induction by a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)), and inhibition by epidermal growth factor in GH3 cells.

A dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP), can induce dopaminergic denervation and Parkinsonism in humans. The active metabolite of MPTP is the 1-methyl-4-phenylpyridinium ion (MPP(+)). Previously we reported that MPP(+) is incorporated via the dopamine transport system and causes delayed cell death in GH3 cells, a clonal strain from the rat anterior pituitary. In this study, we investigated whether MPP(+) induces apoptosis. GH3 cells cultured with MPP(+) exhibited DNA laddering and fragmentation in a time- and concentration-dependent manner. The effect of MPP(+) was inhibited in GH3 cells treated with a pan-caspase inhibitor (100 microM ZVAD-fmk), an antioxidant (25 mM N-acetyl-l-cysteine), or epidermal growth factor (EGF; 50 ng/mL). Because EGF stimulated tyrosine phosphorylation of the EGF receptor and tyrphostin AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline; 5 microM, a specific inhibitor of EGF receptor kinase] abolished EGF inhibition, involvement of EGF receptor kinase is assumed. Protein kinase C-dependent processes and Bcl-2 protein expression were shown not to be involved in EGF inhibition. MPP(+) increased cytochrome c immunoreactivity in cytosolic fractions in GH3 cells. The addition of 200 microM MPP(+) to isolated mitochondrial fractions from GH3 cells stimulated the release of a 13-kDa protein that cross-reacted with anti-cytochrome c antibody. The release was inhibited in EGF-treated GH3 cells. Our findings demonstrated that (i) MPP(+) induces apoptosis of GH3 cells via cytochrome c release and caspase activation, and (ii) apoptosis by MPP(+) can be blocked by N-acetyl-l-cysteine or EGF treatment.

Possible involvement of cytosolic phospholipase A(2) in cell death induced by 1-methyl-4-phenylpyridinium ion, a dopaminergic neurotoxin, in GH3 cells.

Previously we reported that 1-methyl-4-phenylpyridinium ion (MPP(+)), a dopaminergic neurotoxin, induced apoptosis of GH3 cells established from rat anterior pituitary. In the present study, the role of MPP(+) along with that of other apoptotic factors such as Ca(2+) and H(2)O(2) in cell death was examined. Ionomycin induced DNA fragmentation and lactate dehydrogenase (LDH) leakage in GH3 cells. H(2)O(2) also induced LDH leakage. Co-addition of MPP(+), in conditions where MPP(+) had no effect by itself, enhanced ionomycin- and H(2)O(2)-induced cell death. Because the stimulation of phospholipase A(2) (PLA(2)) causing arachidonic acid (AA) release has been proposed to be involved in neuronal cell death, the effect of MPP(+) on AA release in GH3 cells was investigated. MPP(+) treatment for 8 h enhanced ionomycin- and H(2)O(2)-stimulated AA release mediated by activation of cytosolic PLA(2) in a concentration-dependent manner, although MPP(+) by itself had no effect on AA release. An inhibitor of cytosolic PLA(2) inhibited MPP(+)-induced cell death. These findings suggest a synergistic effect of MPP(+) on Ca(2+)- and H(2)O(2)-induced cell death, and the involvement of cytosolic PLA(2) activation in MPP(+)-induced cell death in GH3 cells. Pretreatment with a caspase inhibitor or EGF did not modify the ionomycin- or H(2)O(2)-induced AA release, or enhancement by MPP(+), but the pretreatment inhibited the cell death in the presence and absence of MPP(+). The involvement of caspase(s) on activation of PLA(2) by MPP(+) was excluded, and EGF inhibited MPP(+)-induced cell death downstream of the AA release.

Inhibition of hydrogen peroxide-induced apoptosis but not arachidonic acid release in GH3 cell by EGF.

Reactive oxygen species (ROS) and arachidonic acid (AA) can both function as extra- and intra-cellular messengers to regulate various cell functions including cell death. The effect of ROS on phospholipase A2 (PLA2) activity and/or AA release has not been extensively studied in neuronal cells. In this study, we investigated the effects of H2O2 on AA release and apoptosis in GH3 cells, a clonal strain from rat anterior pituitary. Incubation with H2O2 for 1 h stimulated [3H]AA release in a concentration-dependent manner from prelabeled GH3 cells. [3H]AA release was inhibited by arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic PLA2, and cytosolic PLA2 protein with a molecular mass of 100 kDa was detected by immunoblotting. Culture with 0.2 mM H2O2 and 30 microM AA for 24 h induced lactate dehydrogenase (LDH) leakage, DNA laddering and DNA fragmentation in GH3 cells. In GH3 cells pretreated with EGF (50 ng/ml) for 24 h, LDH leakage and DNA fragmentation by H2O2 and AA were inhibited, although H2O2-induced [3H]AA release was not modified. Mastoparan, a wasp venom peptide, induced [3H]AA release and cell death in GH3 cells. Neither effect of mastoparan was inhibited by EGF treatment. These findings suggest that (1) H2O2 stimulates AA release via activation of cytosolic PLA2, (2) H2O2 and AA induce apoptotic death of GH3 cells and (3) treatment with EGF protects H2O2- and AA-, but not mastoparan-, induced GH3 cell death.

[Successful treatment with donor leukocyte transfusion followed by interferon-alpha in a patient with relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation].

A 44-year-old man with CML in chronic phase was admitted for BMT from an HLA-identical sibling. Ph positive cells were undetectable at 3 and 7 months after BMT but became detectable by cytogenetic analysis of bone marrow aspirates at 12 months after BMT. He was treated with IFN-alpha (6 million units/day, 3 times a week) without apparent effect. Donor leukocyte transfusion (DLT) was performed four times between 20 months and 23 months after BMT, transfusing 3.4 x 10(8) mononuclear cells/kg. However, leukocytosis appeared and the NAP score declined at 25 months after BMT. FISH analysis revealed an increase in bcr-abl positive cells. IFN-alpha was restarted using the same schedule at 26 months after BMT. Three months after restarting IFN-alpha, the leukocyte count fell to the normal range, NAP score increased to a normal level, and bcr-abl positive cells decreased markedly. He has remained in hematological and cytogenetic remission for 20 months, and bcr-abl chimeric mRNA remained undetectable by PCR. These results suggest that CML which does not respond to DLT may be cured by subsequent IFN-alpha therapy, possibly by inducing anti-leukemia immune responses.

Death by a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+) and protection by EGF in GH3 cells.

In this study we investigated the uptake and effect of a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+) on a clonal strain, GH3 cells, established from rat anterior pituitary. Although the level was very low compared with that in PC12 cells, a clonal rat pheochromocytoma cell line, there was a detectable amount of tyrosine hydroxylase protein in GH3 cells. The levels of monoamines including dopamine in GH3 cells were also very low compared with those in PC12 cells. [3H]MPP+ was incorporated to GH3 cells in a concentration-dependent manner and the uptake was inhibited by nomifensine, an inhibitor of dopamine transporter. Addition of 200 microM MPP+ stimulated the leakage of lactate dehydrogenase (LDH) after a lag of 24 h. Pretreatment with 50 ng/ml of epidermal growth factor (EGF), but not nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), protected against MPP+-induced cell death. These findings show that: (1) MPP+ uptake to GH3 cells was via an effective dopamine transport system and causes delayed cell death, and (2) EGF protects against MPP+-induced cell death. A possible role for GH3 cells as dopaminergic neurons is discussed.

Diagnostic and biological significance of sideroblastic erythropoiesis in the myelodysplastic syndromes.

We retrospectively reviewed clinical and hematologic features of nine patients with acquired idiopathic sideroblastic anemia (AISA). Seven of them had ringed sideroblasts (RS) more than 15% of marrow nucleated cells. RS persisted in the marrow even in the remaining two patients who had a relatively low marrow erythroblasts despite RS ranging from 1/4 to half of the marrow erythroid series. However, RS declined in proportion in another two patients of the nine whose disease progressed to refractory anemia with excess of blasts (RAEB), although a high proportion of RS reappeared in one patient at the time of relapse following allogeneic marrow transplantation. A similar decline of RS concomitant with disease progression was also seen in seven additional patients with RAEB or RAEB in transformation (RAEB-t) with sideroblastic erythropoiesis. Cytogenetic abnormalities, although rare initially, became detectable either at the time of disease progression or at the worsening of anemia in AISA. These observations suggest that the majority of AISA fall in the category of myelodysplasia, and that a progressive decline in RS is part of the natural history of myelodysplasia. Closer follow-up of the proportion of RS in patients with AISA is warranted to better understand its biologic significance.

A 5-year retrospective analysis of alpha-interferon usage.

Alpha interferon is an immune modulator used in the treatment of hematologic malignancies and immunosuppressive diseases. While many of the clinical indications for interferon have been well described and are FDA-approved, a large number of clinical uses of interferon are being developed. This study evaluated the appropriateness, efficacy, and safety of interferon therapy at our institution from 1987 to 1991. Data were collected by chart review. Response rates of patients in this hospital were compared to those published in the literature. Twenty-six patients were prescribed alpha interferon. Ten patients (38%) demonstrated a partial response, the highest responses seen in Hairy cell leukemia (67%) and chronic myelogenous leukemia (57%). Response rates for each disease compared favorably to those predicted from the literature. Twelve patients (46%) demonstrated intolerance. Overall five patients (19%) remain on therapy. While interferon appears to be moderately effective in certain diseases, intolerance to interferon seems to be the major limiting factor to its clinical application.

[Complete remission in acute myeloblastic leukemia (M0) after treatment with rhG-CSF].

A 57-year-old man was admitted because of fever and night sweat. The bone marrow was hypercellular with 86.4% blast cells. The diagnosis of AML (M0) was made, because the blast cells were negative for peroxidase stain and had CD13 and no lymphoid antigens in marker analysis. The patient was treated with BH-AC.TMP, BH-AC.MVP and low dose Ara-C without any hematological improvement, and even additional treatment with medium dose Ara-C resulted in 66.4% blast cells in the bone marrow. Subsequent administration of rhG-CSF (150 micrograms/day) by continuous intravenous infusion resulted in the decrease of the blast cells in the bone marrow to a level that was evaluated as complete remission. He remains in complete hematological remission at present. As shown in this case, rhG-CSF might be an effective agent for the treatment of AML, even if the mechanism of its effectiveness is unclear at present. Further clinical studies should will supply useful information to analyze the pathophysiology of AML.