Carbachol, but Not Norepinephrine, NMDA, lonomycin, Ouabain, or Phorbol Myristate Acetate, Increases Inositol 1,3,4,5-Tetrakisphosphate Accumulation in Rat Brain Cortical Slices

Abstract: lonomycin, a Ca²⁺ ionophore, stimulated phosphoinositide breakdown in rat brain cortical slices incubated in the presence of 1.2 m M Ca²⁺, but, unlike muscarinic cholinergic stimulation, it had little effect on inositol 1,3,4,5-tetrakisphosphate accumulation. However, at 2 min, the increase in inositol 1,4,5-trisphosphate due to 10 μ M ionomycin was equivalent to that seen with 1μ M carbachol. Phorbol 12-myristate 13-acetate or high K⁺ (30 μ M ) increased inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5-tetrakisphosphate accumulation. The stimulation of inositol 1,4,5-trisphosphate accumulation due to ionomycin, unlike that seen with carbachol, was abolished in buffer containing 0.2 μ M Ca²⁺. The increase in inositol 1,3,4,5-tetrakisphosphate accumulation in brain slices due to 1 μ M carbachol ranged from 55 to 68% of that for inositol 1,4,5-trisphosphate. Norepinephrine, NMDA, veratridine, and ouabain also increased inositol 1,4,5-tris-phosphate, but had minimal effects on inositol 1,3,4,5-tetrakisphosphate accumulation. These results suggest that there is something unique about the stimulation of inositol 1,3,4,5-tetrakisphosphate accumulation by carbachol, which is also the only one of these agents that is able to activate phosphoinositidase Cβ, in isolated rat brain membranes.     

The effects of benzyladenine, cycloheximide, and cordycepin on wilting-induced abscisic Acid and proline accumulations and abscisic Acid- and salt-induced proline accumulation in barley leaves

Benzyladenine inhibits proline accumulation in wilted, abscisic acid (ABA)-treated, and salt-shocked barley leaves. It does not affect ABA accumulation or disappearance in wilted leaves. Inhibition of proline accumulation in salt-shocked leaves was observed both when benzyladenine was added at the beginning of or after salt treatment. Cycloheximide (CHX) and cordycepin inhibited both ABA and proline accumulations in wilted barley leaves and proline accumulation in ABA-treated leaves. In salt-shocked leaves, cordycepin inhibited proline accumulation when added after salt treatment but before proline began to accumulate but not when added after the onset of proline accumulation. CHX delayed the accumulation of proline in salt-shocked leaves but, after a period of time, proline accumulated in the CHX-treated leaves at rates comparable to the salt-treated control. This delay and subsequent accumulation was observed when CHX was added before, during, and after salt treatment. However, the earlier in the salt treatment period that CHX was given, the longer was the observed delay. These results are interpreted to indicate that gene activation is involved in proline accumulation in response to wilting, to ABA, and to salt in barley leaves. This gene activation is in addition to the gene activation that is required for ABA accumulation in wilted leaves. If ABA accumulation is required for proline accumulation in wilted barley leaves, then two sets of gene activation are involved in wilting-induced proline accumulation. All of our results are consistent with this possibility but do not prove it. The inhibition of proline accumulation by benzyladenine is probably neither due to an effect on gene activation nor to an effect on the ABA level.     

Nutrient balance and P,K, Ca,Mg, S and B accumulation in a Norway spruce stand following ammonium sulphate application,fertigation, irrigation,drought and N-free-fertilisation

The nutrient balance and above ground accumulation of macro nutrients, except for N, resulting from improved or deteriorated availability of water and nutrients in a 25 year old Norway spruce stand in SW Sweden is presented. The site and the productivity of the stand is typical for the area. Treatment include irrigation (I); artificial drought (D1); ammonium sulphate addition (NS); N-free-fertilisation (V) and irrigation with liquid fertilisers including a complete set of nutrients according to the Ingestad principle (IF). At start of the experiment the stand contained 86.5 t dry mass, 342 kg N, 33 kg P, 142 kg K, 172 kg Ca, 36 kg Mg and 34 kg S ha^-1. Enhanced accumulation vs control of S was seen in the NS and IF treatments. In the V and IF treatments P accumulation was 7–9 times higher and Ca and Mg, 2–4 times higher compared to the control. K accumulation was increased for the IF treatment. B that accumulated in the needles was decreased in the NS and D1 treatments and increased in the IF and V treatments, as compared to the control. The gross accumulation of nutrients relative to the amounts added was in the IF and V treatments 56 and 47% for P, 40 and 64% for K, 40 and 24% for Mg and, 22 and 8% for S, respectively. We conclude that application with N-free fertilizer, Skogvital (V), including macro nutrients and essential micro nutrients, results in a fast and efficient accumulation above ground of P, K, Ca, Mg and B. The treatment is efficient when aiming at restoring nutrient imbalances in Norway spruce. Application with ammonium sulphate at a rate of 5–6 times higher than the current deposition of N and S did not lead to decreased accumulation above ground of any of the macro nutrients P, K, Ca or Mg. The accumulation of B, however, was significantly reduced. Results from this and other studies indicate that today, N alone, generally is a growth limiting nutrient for Norway spruce in Southern Sweden.     

Modification of lymphokine-activated killer cell accumulation into tumor sites by chemotherapy, local irradiation, or splenectomy

The effects of chemotherapy or local irradiation on lymphokine-activated killer (LAK) cell accumulation into tumor sites were investigated. Lymphokine-activated killer cells labeled with 111In-oxine were injected into the caudal vein of C57BL/6 mice that had been previously transplanted with 3LL cancer. An adoptive transfer of LAK cells was carried out 4 days after treatments. Twenty-four hours after the transfer, tumor tissues were excised, and the accumulation of labeled LAK cells in the tumor was measured. In two different experiments, LAK cell accumulation in tumor in the nontreated group was 2.15% and 1.58% of the administered dose per gram of tissue. The accumulation in the groups of mice treated with cyclophosphamide, nimustine hydrochloride, or Adriamycin increased fourfold (7.38% dose/g, 6.61% dose/g), threefold (6.47% dose/g) and twofold (4.46% dose/g), respectively, as compared with the nontreated group. These agents induced significant tumor regression. In the group treated with bleomycin, which showed no significant effect on tumor growth, LAK cell accumulation in tumor remained unaltered (1.57% dose/g). However, the group treated with local irradiation, which induced significant tumor reduction, showed no increase in LAK cell accumulation into tumors. These results suggest that some antitumor drugs enhance LAK cell accumulation into tumor sites and that this increase is due to tumor modification by antitumor drugs.     

水分胁迫对蒺藜光合作用、生物量和药材质量的影响

通过盆栽试验,分析了水分胁迫对蒺藜光合特性、生物量形成和主要药用成分积累的影响.结果表明:水分胁迫改变了蒺藜的光合日进程,使蒺藜的净光合速率和蒸腾速率显著降低,植株光合能力减弱,同化能力下降,最终导致干物质积累受阻,生物量降低.但水分胁迫促进了蒺藜总皂苷类物质积累.灌水量为150和450 mm处理下,蒺藜总皂苷含量分别为13.26和12.47 mg·g^-1,与其余处理(250、350 mm)总皂苷含量差异显著.总皂苷和总黄酮类物质动态积累趋势与生物量积累趋势相反,生物量积累高峰期,次生代谢产物含量反而降低生物量积累受外界环境因子的直接影响较大,而次生代谢产物除受外界环境因子的影响外,还受到生物量积累等内部因素影响.     

Effects of laxative and N-acetylcysteine on mucus accumulation, bacterial load, transit, and inflammation in the cystic fibrosis mouse small intestine

The accumulation of mucus in affected organs is characteristic of cystic fibrosis (CF). The CF mouse small intestine has dramatic mucus accumulation and exhibits slower interdigestive intestinal transit. These factors are proposed to play cooperative roles that foster small intestinal bacterial overgrowth (SIBO) and contribute to the innate immune response of the CF intestine. It was hypothesized that decreasing the mucus accumulation would reduce SIBO and might improve other aspects of the CF intestinal phenotype. To test this, solid chow-fed CF mice were treated with an osmotic laxative to improve gut hydration or liquid-fed mice were treated orally with N-acetylcysteine (NAC) to break mucin disulfide bonds. Treatment with laxative or NAC reduced mucus accumulation by 43% and 50%, respectively, as measured histologically as dilation of the intestinal crypts. Laxative and NAC also reduced bacterial overgrowth in the CF intestine by 92% and 63%, respectively. Treatment with laxative normalized small intestinal transit in CF mice, whereas NAC did not. The expression of innate immune response-related genes was significantly reduced in laxative-treated CF mice, whereas there was no significant effect in NAC-treated CF mice. In summary, laxative and NAC treatments of CF mice reduced mucus accumulation to a similar extent, but laxative was more effective than NAC at reducing bacterial load. Eradication of bacterial overgrowth by laxative treatment was associated with normalized intestinal transit and a reduction in the innate immune response. These results suggest that both mucus accumulation and slowed interdigestive small intestinal transit contribute to SIBO in the CF intestine.     

Nutrient limitation of phytoplankton growth in Waquoit Bay, Massachusetts, USA: a nutrient enrichment study

We conducted nutrient enrichment experiments and field sampling to address three questions: (1) is there nutrient limitation of phytoplankton accumulation within an estuary whose waters are exposed to relatively high nitrogen loading rates, (2) where in the salinity gradient from fresh to seawater (0 to 32‰) is there a shift from phosphorus to nitrogen limitation of phytoplankton accumulation, and (3) is there a seasonal shift in limiting function of phosphorus and nitrogen anywhere in the estuarine gradient. Nitrogen and phosphorus enrichment experiments in the Childs River, an estuary of Waquoit Bay, Massachusetts, USA, showed that the accumulation of phytoplankton biomass in brackish and saline water was limited by supply of nitrate during warm months. The effects of enrichment were less evident in fresh water, with short-lived responses to phosphate enrichment. There was no specific point along the salinity gradient where there was a shift from phosphorus- to nitrogen-limited phytoplankton accumulation; rather, the relative importance of nitrogen and phosphorus changed along the salinity gradient in the estuary and with season of the year. There was no response to nutrient additions during the colder months, suggesting that some seasonally-varying factor, such as light, temperature or a physiological mechanism, restricted phytoplankton accumulation during months other than May-Aug. There was only slight evidence of a seasonal shift between nitrogen- and phosphorus-limitation of chlorophyll accumulation. Phytoplankton populations in nutrient-rich estuaries with short flushing times grow fast, but at the same time the cells may be advected out of the estuaries while still rapidly dividing, thereby providing an important subsidy to production in nearby deeper waters. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chitinase induced by jasmonic acid,methyl jasmonate,ethylene and protein phosphatase inhibitors in rice

Chitinase is a pathogenesis-related protein that hydrolyzes chitin, a major component of fungal cell walls. Two-week-old rice seedling leaf, leaf sheath and root tissues responded to an exogenous treatment by jasmonic acid (JA) with induction of the chitinases as determined by immunoblot analysis using an anti-endochitinase antibody. Induced accumulation of these chitinases was observed within 24 to 48 h in the leaf sheaths, leaves and roots. Besides, ethylene generator ethephon and abiotic stressor copper could also induce chitinases accumulation among various plant hormones and stress agents examined. Cycloheximide effectively blocked their accumulation by JA, suggesting that de novo protein synthesis is required. Partial blockage of the induced accumulation of chitinases by NADPH oxidase inhibitor and free radical scavengers suggested involvement of reactive oxygen species. Moreover, induced accumulation of these chitinases also by methyl jasmonate and certain protein phosphatase inhibitors indicated their potential importance and wider role in rice seedlings.     

Germanium and silver resistance, accumulation, and toxicity in microorganisms.

Germanium is an inert metal with no known biological function in prokaryotic or eukaryotic organisms. Its toxicity is low compared to that of silver. Germanium is accumulated in certain bacterial strains by either energy-independent passive binding or an energy-dependent mechanism. Little is known about the molecular aspects of silver resistance, toxicity, and accumulation in bacterial strains. This is surprising because silver has been used as an antimicrobial agent in the medical field for centuries. It is likely that silver ions are excluded (resulting in decreased silver accumulation) from certain bacterial strains or immobilized intracellularly to prevent toxic effects from being exerted. These mechanisms of silver resistance have not been fully elucidated. This review examines the toxicity and accumulation of germanium and silver in selected microbial species. In addition, resistance mechanisms to these biologically nonessential metals is discussed, with more emphasis placed on silver-resistant bacteria due to the knowledge available.     

The synthesis of astaxanthin esters,independent of the formation of cysts,highly correlates with the synthesis of fatty acids in Haematococcus pluvialis

The compositions and contents of astaxanthin esters and fatty acids in four types of Haematococcus pluvialis cells were studied by HPLC and GC-MS. Results showed that the synthesis and accumulation of astaxanthin was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of fatty acids, though it is an well known phenomenon that the accumulation of astaxanthin is usually accompanied by the formation of cyst. The red cysts contain more than 30% of fatty acids, with 81% of the unsaturated fatty acids. Taken together, besides a resource of astaxanthin, H. pluvialis would be a good resource of valuable fatty acids.     

Infection, propagation, distribution and stability of plant virus in hairy root cultures

Nicotiana benthamiana hairy root cultures were infected with tobacco mosaic virus (TMV) and used for in vitro plant virus propagation. The roots were infected with TMV by addition of virus to the medium at the same time as root inoculation. Viral accumulation in the biomass was 7-11-fold greater when the initial infection was carried out in B5 medium rather than sodium phosphate buffer; virus accumulation also increased with increasing viral inoculum concentration. The amount of TMV accumulated in the biomass was similar when virus was retained in the medium for the duration of the cultures and when the inoculum virus was removed 23h after addition to the roots. In roots with established infections, the concentration of virus remained relatively constant and did not increase with further root growth. The distribution of virus within individual root mats harvested from shake flasks was not uniform; there was also significant variability in viral accumulation between replicate hairy root cultures. The picture that emerges from this work is that in vitro viral accumulation in hairy root cultures depends strongly on the viral inoculum concentration applied and the initial level of primary infection achieved, even though primary infection by external virus occurs mainly within only the first few hours of exposure to the biomass and is followed by substantial secondary infection by viral progeny within the root tissue.     

Characteristics of Cd uptake, translocation and accumulation in a novel Cd-accumulating tree, star fruit (Averrhoa carambola L., Oxalidaceae)

Cadmium (Cd) accumulation by terrestrial higher plants is an intriguing phenomenon that may be exploited for phytoextraction of Cd-contaminated soils. Characterizing the physiological processes responsible for elevated concentrations of Cd in shoots is a first step towards a comprehensive understanding of the mechanisms underlying Cd accumulation in plants and may eventually improve the efficiency of phytoextraction. Woody species that can accumulate Cd have been recently recommended as good candidates for phytoextraction of Cd-contaminated soils. However, little is known about the mechanisms of Cd accumulation by woody species. In an attempt to understand the physiological processes contributing to Cd accumulation in woody species, Cd uptake and translocation by a novel tropical Cd-accumulating tree, star fruit (Averrhoa carambola) were characterized and compared with those of a non-Cd-accumulating tree (Clausena lansium). Our results showed that A. carambola had higher Cd uptake and root-to-shoot translocation efficiencies than C. lansium, which might account for its greater Cd-accumulating capacity. Furthermore, Cd accumulation by A. carambola was not significantly affected by zinc (Zn), whereas Zn accumulation was greatly lowered by Cd. This phenomenon could not be fully explained by a simple competition between Cd²⁺ and Zn²⁺, implying the existence of a transport system with a preference for Cd over Zn. Collectively, our results indicate that A. carambola has noteworthy physiological traits associated with accumulation of Cd to high levels.     

Genotypic-dependent effects of N fertilizer,glutathione, silicon,zinc, and selenium on proteomic profiles,amino acid contents,and quality of rice genotypes with contrasting grain Cd accumulation

Soil heavy metal (HM) contamination has posed a serious problem for safe food production. For restricting the translocation of HM into grain, many proteins were regulated to involve in the process. To identify these proteins, 2D-based proteomic analysis was carried out using different rice genotypes with distinct Cd accumulation in grains and as affected by an alleviating regulator (AR) in field experiments. AR application improved grain quality, with increased contents in Glu, Cys, His, Pro, and protein. Twenty-six low-grain HM accumulation-associated protein species were identified and categorized as physiological functions via two-dimensional gel electrophoresis (2DE) and mass spectrometry. Among these proteins, 8, 9, and 9 proteins exhibited higher accumulation, lower accumulation, and unchanged accumulation, respectively, in Xiushui817 (low accumulator) vs R8097 (high accumulator) under control conditions but showed differential accumulation patterns after AR application. These proteins included sucrose synthase 3, alanine aminotransferase, glutelin, cupin family protein, and zinc finger CCCH domain-containing protein 32. The differential expression of these protein species might contribute to decreased HM accumulation in grain via decreasing the protein accumulation which had high affinity to HM or regulating energy metabolism and signal transduction. Our findings provide valuable insights into the mechanisms of low-grain HM accumulation in rice and possible utilization of candidate protein species in developing low-grain HM accumulation genotypes.     

A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells

HIV protease inhibitors (HIV-PIs) are key components of highly active antiretroviral therapy, but they have been associated with adverse side effects, including partial lipodystrophy and metabolic syndrome. We recently demonstrated that a commonly used HIV-PI, lopinavir, inhibits ZMPSTE24, thereby blocking lamin A biogenesis and leading to an accumulation of prelamin A. ZMPSTE24 deficiency in humans causes an accumulation of prelamin A and leads to lipodystrophy and other disease phenotypes. Thus, an accumulation of prelamin A in the setting of HIV-PIs represents a plausible mechanism for some drug side effects. Here we show, with metabolic labeling studies, that lopinavir leads to the accumulation of the farnesylated form of prelamin A. We also tested whether a new and chemically distinct HIV-PI, darunavir, inhibits ZMPSTE24. We found that darunavir does not inhibit the biochemical activity of ZMPSTE24, nor does it lead to an accumulation of farnesyl-prelamin A in cells. This property of darunavir is potentially attractive. However, all HIV-PIs, including darunavir, are generally administered with ritonavir, an HIV-PI that is used to block the metabolism of other HIV-PIs. Ritonavir, like lopinavir, inhibits ZMPSTE24 and leads to an accumulation of prelamin A.     

Withaferin A Induces Proteasome Inhibition,Endoplasmic Reticulum Stress,the Heat Shock Response and Acquisition of Thermotolerance

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.     

Accumulation of arsenate, phosphate, and aspartate by Sreptococcus faecalis.

Uptake of arsenate and phosphate by Streptococcus faecalis 9790 is strictly dependent on concurrent energy metabolism and essentially unidirectional. targinine supports uptake only in presence of glycerol or related substances; glycerol is not directly involved in transport but depletes the cellular orthophosphate pool and thus relieves feedback inhibition of transport. Uptake of phosphate and arsenate is stimulated by K+ and by other permeant cations. The results suggest that electroneutrality is preserved by compensatory movement of either H+ or OH minus. Ionophores and N,N'-dicyclohexylcarbodiimide, which prevent establishment of a proton motive force, block the accumulation of thiomethylgalactoside and of threonine but not that of arsenate or phosphate. We conclude that arsenate accumulation requires adenosine 5'-triphosphate but is not driven by the proton-motive force. However, conditions and reagents that lower the cytoplasmic pH do inhibit accumulation of arsenate and phosphate, suggesting that uptake depends on the capacity of the cells to maintain a neutral or alkaline cytoplasm. We therefore propose that phosphate accumulation is an electroneutral exchange for OH driven by adenosine 5'-triphosphate or by a metabolite thereof. Accumulation of aspartate and glutamate also requires adenosine 5'-triphosphate but not the proton-motive force and may involve a similar mechanism.     

Mechanism of D-cycloserine action: transport systems for D-alanine, D-cycloserine, L-alanine, and glycine

The accumulation of d-alanine, l-alanine, glycine, and d-cycloserine in Escherichia coli was found to be mediated by at least two transport systems. The systems for d-alanine and glycine are related, and are separate from that involved in the accumulation of l-alanine. d-Cycloserine appears to be primarily transported by the d-alanine-glycine system. The accumulation of d-alanine, glycine, and d-cycloserine was characterized by two line segments in the Lineweaver-Burk analysis, whereas the accumulation of l-alanine was characterized by a single line segment. d-Cycloserine was an effective inhibitor of glycine and d-alanine accumulation, and l-cycloserine was an effective inhibitor of l-alanine transport. The systems were further differentiated by effects of azide, enhancement under various growth conditions, and additional inhibitor studies. Since the primary access of d-cycloserine in E. coli is via the d-alanine-glycine system, glycine might be expected to be a better antagonist of d-cycloserine inhibition than l-alanine. Glycine and d-alanine at 10(-5)m antagonized the effect of d-cycloserine in E. coli, whereas this concentration of l-alanine had no effect.     

Water stress, ammonium, and leaf senescence in detached rice leaves

Ammonium accumulation in relation to water stress-promoted senescence of detached rice leaves was investigated. The effect of water stress on the senescence of detached rice leaves is associated with the accumulation of ammonium. The accumulation of ammonium in detached rice leaves by water stress is attributed to a decrease in glutamine synthetase activity. Ammonium accumulation in detached rice leaves, induced by water stress, was accompanied by an increase in tissue sensitivity to ethylene which, in turn, accelerated leaf senescence.     

An antiapoptotic protein, c-FLIPL, directly binds to MKK7 and inhibits the JNK pathway

Inhibition of NF-kappaB activation increases susceptibility to tumor necrosis factor (TNF)alpha-induced cell death, concurrent with caspases and prolonged c-Jun N-terminal kinase (JNK) activation, and reactive oxygen species (ROS) accumulation. However, the detailed mechanisms are unclear. Here we show that cellular FLICE-inhibitory protein (c-FLIP) is rapidly lost in NF-kappaB activation-deficient, but not wild-type fibroblasts upon TNFalpha stimulation, indicating that NF-kappaB normally maintains the cellular levels of c-FLIP. The ectopic expression of the long form of c-FLIP (c-FLIPL) inhibits TNFalpha-induced prolonged JNK activation and ROS accumulation in NF-kappaB activation-deficient fibroblasts. Conversely, TNFalpha induces prolonged JNK activation and ROS accumulation in c-Flip-/- fibroblasts. Moreover, c-FLIPL directly interacts with a JNK activator, MAP kinase kinase (MKK)7, in a TNFalpha-dependent manner and inhibits the interactions of MKK7 with MAP/ERK kinase kinase 1, apoptosis-signal-regulating kinase 1, and TGFbeta-activated kinase 1. This stimuli-dependent interaction of c-FLIPL with MKK7 might selectively suppress the prolonged phase of JNK activation. Taken that ROS promote JNK activation and activation of the JNK pathway may promote ROS accumulation, c-FLIPL might block this positive feedback loop, thereby suppressing ROS accumulation.