Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors

Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli.     

Elevated Accumulation of Proline in NaCl-Adapted Tobacco Cells Is Not Due to Altered Delta-Pyrroline-5-Carboxylate Reductase

Tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that are adapted to 428 millimolar NaCl accumulate proline mainly due to increased synthesis from glutamate. These cells were used to evaluate the possible role of Δ¹-pyrroline-5-carboxylate reductase in the regulation of proline biosynthesis. No increase in the specific activity of Δ¹-pyrroline-5-carboxylate reductase in crude extracts throughout the growth cycle was observed in NaCl-adapted cells compared to unadapted cells. The enzyme from both cell types was purified extensively. On the basis of affinity for the substrates NADPH, NADH, and Δ¹-pyrroline-5-carboxylate, pH profiles, chromatographic behavior during purification, and electrophoretic mobility of the native enzyme, the activities of the enzyme from the two sources were similar. These data suggest that the NaCl-dependent regulation of proline synthesis in tobacco cells does not involve induction of pyrroline-5-carboxylate isozymes or changes in its kinetic properties.     

Nitric Oxide Synthase Inhibitors Do Not Attenuate Diacylglycerol or Monoacylglycerol Lipase Activities in Synaptoneurosomes

Neuron-enriched cultures and synaptoneurosomal fractions from 10 day-old rat brain contain diacylglycerol and monoacylglycerol lipase activities. Glutamate and its analogs stimulate the activities of diacylglycerol and monoacylglycerol lipases in a time-and dose-dependent manner. Stimulation of diacylglycerol and monoacylglycerol lipases by glutamate or NMDA can be blocked by MK-801 (non-competitive antagonist). Nitro L-arginine methyl ester and L-methylarginine have no effect on glutamate stimulated activities of diacylglycerol and monoacylglycerol lipases. Our studies suggest that synaptoneurosomal preparations from young rat brain are useful for obtaining important information on signal transduction.     

拟南芥乙酰羟酸合成酶(AHAS)点突变原核表达与活性测定

拟南芥乙酰羟酸合成酶(AHAS)参与支链氨基酸合成。为考察AHAS不同结构域对支链氨基酸合成的影响,分别对其大小亚基上特定位点进行点突变后进行原核表达,体外重组后对其全酶活性进行测定,并对其终端产物之一——缬氨酸对AHAS全酶活性的影响进行探讨。结果显示:AHAS小亚基G88D突变将解除其终端产物的反馈抑制作用,而大亚基E305D与E482D的突变降低AHAS全酶活性,且2种不同突变大亚基对AHAS全酶活性影响存在差异。AHAS大亚基E482D突变较E305D突变影响更大。研究结果表明:AHAS大小亚基间存在着相互作用,且大小亚基不同结构域突变对AHAS全酶活性具有不同的影响。     

Sphingomyelin Synthase 2, but Not Sphingomyelin Synthase 1, Is Involved in HIV-1 Envelope-mediated Membrane Fusion

Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was ∼5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor·co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.     

GUVs Melt Like LUVs: The Large Heat Capacity of MLVs Is Not Due to Large Size or Small Curvature

The excess heat capacity functions (ΔC_p) associated with the main phase transition of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) are very different. Two explanations are possible. First, the difference in vesicle size (curvature) results in different gel-fluid interactions in the membrane; those interactions have a large effect on the cooperativity of the phase transition. Second, there is communication between the bilayers in an MLV when they undergo the gel-fluid transition; this communication results in thermodynamic coupling of the phase transitions of the bilayers in the MLV and, consequently, in an apparent increase in the cooperativity of the transition. To test these hypotheses, differential scanning calorimetry was performed on giant unilamellar vesicles (GUVs) of pure dipalmitoylphosphatidylcholine. The ΔC_p curve of GUVs was found to resemble that of the much smaller LUVs. The transition in GUVs and LUVs is much broader (half-width ∼1.5°C) than in MLVs (∼0.1°C). This similarity in GUVs and LUVs indicates that their size has little effect on gel-fluid interactions in the phase transition. The result suggests that coupling between the transitions in the bilayers of an MLV is responsible for their apparent higher cooperativity in melting.     

Charge Heterogeneity of Insulin Fusion Proteins Expressed in Escherichia coli Is Not Due to Proteolytic Degradation

Evaluation of the yield of expression of exogeneous protein in transformed Escherichia coli cells by means of one-dimensional SDS-PAGE often leads to overestimation and miscalculation. For example, it is possible that proteins of similar size comigrate and thus mask the overexpressed product band. Therefore, two-dimensional electrophoresis was used to analyze two types of recombinant fusion proteins, i.e., a β-galactosidase insulin fusion protein and a interleukin II insulin fusion protein, directly after fermentation. We found that production scale expression products show charge and size heterogeneity. The heterogeneous protein spots were characterized by subsequent blotting onto Immobilon membrane and by N-terminal sequencing. Some of the separated spots were either N-terminally blocked or already degraded to some extent. The integrity of the actual product component of the fusion protein was examined with a C-terminus-specific antibody and by Western blot analysis of the 2D gels.     

Heat Killing of Bacillus subtilis Spores in Water Is Not Due to Oxidative Damage

The heat resistance of wild-type spores of Bacillus subtilis or spores (termed α⁻β⁻) lacking DNA protective α/β-type small, acid-soluble spore proteins was not altered by anaerobiosis or high concentrations of the free radical scavenging agents ethanethiol and ethanedithiol. Heat-killed wild-type and α⁻β⁻ spores exhibited no increase in either protein carbonyl content or oxidized bases in DNA. These data strongly suggest that oxidative damage to spore macromolecules does not contribute significantly to spore killing by heat.     

Bilateral Strength Deficit Is Not Neural in Origin; Rather Due to Dynamometer Mechanical Configuration

During maximal contractions, the sum of forces exerted by homonymous muscles unilaterally is typically higher than the sum of forces exerted by the same muscles bilaterally. However, the underlying mechanism(s) of this phenomenon, which is known as the bilateral strength deficit, remain equivocal. One potential factor that has received minimal attention is the contribution of body adjustments to bilateral and unilateral force production. The purpose of this study was to evaluate the plantar-flexors in an innovative dynamometer that permitted the influence of torque from body adjustments to be adapted. Participants were identically positioned between two setup configurations where torques generated from body adjustments were included within the net ankle torque (locked-unit) or independent of the ankle (open-unit). Twenty healthy adult males performed unilateral and bilateral maximal voluntary isometric plantar-flexion contractions using the dynamometer in the open and locked-unit mechanical configurations. While there was a significant bilateral strength deficit in the locked-unit (p = 0.01), it was not evident in the open-unit (p = 0.07). In the locked-unit, unilateral torque was greater than in the open-unit (p<0.001) and this was due to an additional torque from the body since the electromyographic activity of the agonist muscles did not differ between the two setups (p>0.05). This study revealed that the mechanical configuration of the dynamometer and then the body adjustments caused the observation of a bilateral strength deficit.     

Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Not Due to Decarbamylation of the Catalytic Site

An investigation was made of the proposal that the slow inactivation of ribulosebisphosphate carboxylase (Rubisco) activity, which occurs during in vitro assays, is due to decarbamylation of the enzyme. The level of carbamylation was compared with catalytic activity during assay conditions in which activity was both increasing and decreasing. Carbamylation level was measured using the reaction-intermediate analogue 2' -carboxy-D-arabinitol-1, 5-bisphosphate (carboxyarabinitol-P(2)). A dual isotope procedure was used in which [(3)H]carboxyarabinitol-P(2) measured total active sites and (14)CO(2) reported the level of carbamylation. The efficacy of the procedure was verified both in the presence and in the absence of the substrate d-ribulose-1, 5-bisphosphate (ribulose-P(2)). These measurements showed that changes in activity during assays were not correlated with carbamylation status. Inactivation during assays initiated with both fully and partially carbamylated enzyme was not associated with any change in carbamylation level. This implies that the loss of activity during assays is not due to ribulose-P(2) binding and sequestering the E form of the enzyme. Ribulose-P(2) did not appear to alter the equilibrium between carbamylated and uncarbamylated enzyme, but it did slow the rate at which enzyme was both decarbamylated and carbamylated. The most likely explanation for the loss of activity during assays appears to be the sequestration of carbamylated, Mg(2+)-bound active sites by an inhibitor.     

Maitotoxin-Evoked γ-Aminobutyric Acid Release Is Due Not Only to the Opening of Calcium Channels

The effects of maitotoxin (MTX) on endogenous amino acid release were tested on highly purified striatal neurons differentiated in primary culture. MTX induced a large and concentration-dependent release of gamma-aminobutyric acid (GABA). This effect was abolished when experiments were performed in the absence of external Ca2+, and restored when Ca2+ ions were added after removing the MTX-containing Ca2+-free solution. MTX-induced amino acid release was not affected by 1 microM nifedipine and only slightly inhibited by 1 mM Co2+. MTX also induced a massive accumulation of 45Ca2+ in the neurons which, in contrast to the MTX-evoked GABA release, was totally blocked in the presence of 1 mM Co2+. Whereas 500 nM tetrodotoxin was without significant effect, MTX-evoked GABA release was dependent on the presence of external Na+ and sensitive to nipecotic acid, a GABA uptake inhibitor. It is concluded that, on striatal neurons, MTX induced Na+ influx only in the presence of external Ca2+. The increase in cytoplasmic Na+ ions then triggers the release of GABA.     

Apparent Inhibition of Chloroplast Protein Import by Cold Temperatures Is Due to Energetic Considerations Not Membrane Fluidity

The transport of proteins across virtually all types of biological membranes has been reported to be inhibited by low temperatures. Paradoxically, plants are able to acclimate to growth at temperatures below which protein import into chloroplasts is known to be blocked. In examining this incongruity, we made a number of unexpected observations. First, chloroplasts isolated from plants grown at 7/1[deg]C in light/dark and from plants grown at 25[deg]C were able to import proteins with the same efficiency over a temperature range from 5 to 21[deg]C, indicating that no functional adaptation had taken place in the protein import machinery of chloroplasts in these cold-grown plants. Second, chloroplasts from warm-grown plants were able to take up proteins at temperatures as low as 4[deg]C provided that they were illuminated. We determined that light mediates the import process at 5[deg]C by driving ATP synthesis in the stroma, the site of its utilization during protein transport. Direct measurement of the envelope phase transition temperature as well as the activity of the ATP/ADP translocator in the inner envelope membrane at 5 and 25[deg]C demonstrated that the cold block of protein import into chloroplasts observed in vitro is due primarily to energetic considerations and not to decreased membrane fluidity.     

Activation of γ-Aminobutyrate Production by Chloroplastic H2O2 Is Associated with the Oxidative Stress Response

A series of N-substituted aryl and alkyl carbamates (RNHCOOR′; R: aryl, alkyl; R′: aryl, alkyl) was prepared and screened for inhibitory activity toward the germination of oat seeds. The activity of each compound was compared with that of chlorpropham (isopropyl 3-chlorocarbanilate). Some of the synthetic carbamates possessing the N-(phenylthio)methyl group, PhSCH₂NHCOOR´, showed inhibitory activity close or comparable to that of chlorpropham.     

EphA2 Is a Therapy Target in EphA2-Positive Leukemias but Is Not Essential for Normal Hematopoiesis or Leukemia

Members of the Eph family of receptor tyrosine kinases and their membrane bound ephrin ligands have been shown to play critical roles in many developmental processes and more recently have been implicated in both normal and pathological processes in post-embryonic tissues. In particular, expression studies of Eph receptors and limited functional studies have demonstrated a role for the Eph/ephrin system in hematopoiesis and leukemogenesis. In particular, EphA2 was reported on hematopoietic stem cells and stromal cells. There are also reports of EphA2 expression in many different types of malignancies including leukemia, however there is a lack of knowledge in understanding the role of EphA2 in hematopoiesis and leukemogenesis. We explored the role of EphA2 in hematopoiesis by analyzing wild type and EphA2 knockout mice. Mature, differentiated cells, progenitors and hematopoietic stem cells derived from knockout and control mice were analyzed and no significant abnormality was detected. These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis. Comparative studies using EphA2-negative MLL-AF9 leukemias derived from EphA2-knockout animals showed that there was no detectable functional role for EphA2 in the initiation or progression of the leukemic process. However, expression of EphA2 in leukemias initiated by MLL-AF9 suggested that this protein might be a possible therapy target in this type of leukemia. We showed that treatment with EphA2 monoclonal antibody IF7 alone had no effect on tumorigenicity and latency of the MLL-AF9 leukemias, while targeting of EphA2 using EphA2 monoclonal antibody with a radioactive payload significantly impaired the leukemic process. Altogether, these results identify EphA2 as a potential radio-therapeutic target in leukemias with MLL translocation.     

Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation

Because CaMKII is the critical Ca²⁺ sensor that triggers long-term potentiation (LTP), understanding its activation and deactivation is important. A major advance has been the development of a FRET indicator of the conformational state of CaMKII called Camui. Experiments using Camui have demonstrated that the open (active) conformation increases during LTP induction and then decays in tens of seconds, with the major fast component decaying with a time-constant of ~ 6 sec (tau1). Because this decay is faster if autophosphorylation of T286 is prevented (the autophosphorylation prolongs activity by making the enzyme active even after Ca²⁺ falls), it seemed likely that the fast decay is due to the T286 dephosphorylation. To test this interpretation, we studied the effect of phosphatase inhibitors on the single-spine Camui signal evoked by two-photon glutamate uncaging. We applied inhibitors of PP1 and PP2A, two phosphatases that are present at synapses and that have been shown to dephosphorylate CaMKII in vitro. The inhibitors increased the basal Camui activation state, indicating their effectiveness in cells. However, in no case did we find that tau1 was prolonged, contrary to what would be expected if the decay was phosphatase-dependent. This could either mean that decay was due to some unknown phosphatase or that the decay was not due to dephosphorylation. To distinguish between these possibilities, we expressed pseudo-phosphorylated Camui (T286D) (plus additional mutations [T/A] that prevented inhibitory 305/306 phosphorylation). This form had an elevated basal activation state, but was further activated during glutamate uncaging; importantly the activation state decayed with tau1 nearly the same as that of WT Camui. Therefore, the data strongly indicate that tau1 is not due to T286 dephosphorylation. We conclude that, although Camui is an excellent tool for observing CaMKII signaling, further experimentation is needed to determine how CaMKII is turned off by its dephosphorylation.     

Increased Zinc Tolerance in Silene vulgaris (Moench) Garcke Is Not Due to Increased Production of Phytochelatins

The concentration of acid-soluble thiols other than reduced glutathione (SH - GSH) increases in the roots of zinc-sensitive and zinc-tolerant Silene vulgaris (Moench) Garcke after exposure to zinc for 1 to 3 d. The concentration of SH - GSH in the roots is higher in the sensitive plants than in the tolerant ones, both at equal external zinc concentrations and at zinc concentrations causing the same level of root-length growth inhibition. High performance liquid chromatography analyses show that the increase in the concentration of SH - GSH is not only due to the production of phytochelatins, but is also due to an increase in the concentration of cysteine and the production of nonidentified thiols. The cysteine concentration increases equally in the roots of sensitive and tolerant plants. The accumulation of phytochelatins is higher in the roots of the sensitive plants, whereas the chain length distribution of phytochelatins is the same in sensitive and tolerant plants. It is concluded that increased zinc tolerance in S. vulgaris is not due to increased production of phytochelatins.     

The Increase in H2O2-Induced Apoptosis by ADP-Ribosylation Inhibitors Is Related to Cell Blebbing

HN and LN are two phenotypic variants of the U937 monocytic cell line which differ in their basal NAD content; they respond in an opposite way to oxidative stress in the presence of the poly(ADP-ribosyl)polymerase (PARP) inhibitors 3-aminobenzamide (3ABA) and nicotinamide (NA): the inhibitors protect HN cells from stress-induced apoptosis, while they enhance it on LN cells (Coppola et al., 1995, Exp. Cell Res. 221,462-469). These opposite effects are due to two overlapping and contrasting phenomena occurring in LN cells, as shown by the bi-modal response of stressed LN cells to increasing 3ABA doses. Indeed H₂O₂-induced apoptosis is enhanced only at high 3ABA concentrations (i.e., sufficient to inhibit also mono-ADP-ribosylations); lower 3ABA concentrations, which specifically inhibit PARP, also protect LN U937 from stress-induced apoptosis. Unlike HN U937, H₂O₂-induced apoptosis in LN cells is accompanied by cell blebbing. High 3ABA doses strongly enhance blebbing, leading to cellular fragmentation. Blebbing could be blocked by interfering with actin polymerization with cytochalasin B and D: this eliminated the increase in apoptosis due to 3ABA, suggesting that it is indeed the consequence of excess blebbing. This is supported by the unusual finding that in U937 LN stressed in the presence of 3ABA or NA, blebbing, usually a late event in apoptosis, may even precede its onset.