Regulation of NO(3) Influx in Barley : Studies Using NO(3)

Short-term (10 minutes) measurements of plasmalemma NO₃⁻ influx (_oc) into roots of intact barley plants were obtained using ¹³NO₃⁻. In plants grown for 4 days at various NO₃⁻ levels (0.1, 0.2, 0.5 millimolar), _oc was found to be independent of the level of NO₃⁻ pretreatment. Similarly, pretreatment with Cl⁻ had no effect upon plasmalemma ¹³NO₃⁻ influx. Plants grown in the complete absence of ¹³NO₃⁻ (in CaSO₄ solutions) subsequently revealed influx values which were more than 50% lower than for plants grown in NO₃⁻. Based upon the documented effects of NO₃⁻ or Cl⁻ pretreatments on net uptake of NO₃⁻, these observations suggest that negative feedback from vacuolar NO₃⁻ and/or Cl⁻ acts at the tonoplast but not at the plasmalemma. When included in the influx medium, 0.5 millimolar Cl⁻ was without effect upon ¹³NO₃⁻ influx, but NH₄⁺ caused approximately 50% reduction of influx at this concentration.     

Regulation of NO(3) Assimilation by Anion Availability in Excised Soybean Leaves

The regulation of NO₃⁻ assimilation by xylem flux of NO₃⁻ was studied in illuminated excised leaves of soybean (Glycine max L. Merr. cv Kingsoy). The supply of exogenous NO₃⁻ at various concentrations via the transpiration stream indicated that the xylem flux of NO₃⁻ was generally rate-limiting for NO₃⁻ reduction. However, NO₃⁻ assimilation rate was maintained within narrow limits as compared with the variations of the xylem flux of NO₃⁻. This was due to considerable remobilization and assimilation of previously stored endogenous NO₃⁻ at low exogenous NO₃⁻ delivery, and limitation of NO₃⁻ reduction at high xylem flux of NO₃⁻, leading to a significant accumulation of exogenous NO₃⁻. The supply of ¹⁵NO₃⁻ to the leaves via the xylem confirmed the labile nature of the NO₃⁻ storage pool, since its half-time for exchange was close to 10 hours under steady state conditions. When the xylem flux of ¹⁵NO₃⁻ increased, the proportion of the available NO₃⁻ which was reduced decreased similarly from nearly 100% to less than 50% for both endogenous ¹⁴NO₃⁻ and exogenous ¹⁵NO₃⁻. This supports the hypothesis that the assimilatory system does not distinguish between endogenous and exogenous NO₃⁻ and that the limitation of NO₃⁻ reduction affected equally the utilization of NO₃⁻ from both sources. It is proposed that, in the soybean leaf, the NO₃⁻ storage pool is particularly involved in the short-term control of NO₃⁻ reduction. The dynamics of this pool results in a buffering of NO₃⁻ reduction against the variations of the exogenous NO₃⁻ delivery.     

Effect of Oxygen and Malate on NO(3) Inhibition of Nitrogenase in Soybean Nodules

Soybean (Glycine max cv Hodgson) nitrogenase activity (C₂H₂ reduction) in the presence or absence of nitrate was studied at various external O₂ tensions. Nitrogenase activity increased with oxygen partial pressure up to 30 kilopascals, which appeared to be the optimum. A parallel increase in ATP/ADP ratios indicated a limitation of respiration rate by low O₂ tensions in the nodule, and the values found for adenine nucleotide ratios suggested that the nitrogenase activity was limited by the rate of ATP regeneration. In the presence of nitrate, the nitrogenase activity was low and less stimulated by increased pO₂, although the nitrite content per gram of nodules decreased from 0.05 to 0.02 micromole when pO₂ increased from 10 to 30 kilopascals. Therefore, the accumulation of nitrite inside the nodule was probably not the major cause of the inhibition. Instead, inhibition by nitrate could be due to competition for reducing power between nitrate reduction and bacteroid or mitochondrial respiration inside the nodule. This is supported by the observation of decrease in ATP/ADP ratios from 1.65, in absence of nitrate, to 0.93 in the presence of this anion at 30 kilopascals O₂. Furthermore, the inhibition was suppressed by the addition, to the plant nutrient solution, of 15 millimolar l-malate, a carbon substrate that is considered to be the major source of reductant for the bacteroids in the symbiosis.     

Effect of Phloem-Translocated Malate on NO(3) Uptake by Roots of Intact Soybean Plants

In soybean (Glycine max L. Merr. cv Kingsoy), NO₃⁻ assimilation in leaves resulted in production and transport of malate to roots (B Touraine, N Grignon, C Grignon [1988] Plant Physiol 88: 605-612). This paper examines the significance of this phenomenon for the control of NO₃⁻ uptake by roots. The net NO₃⁻ uptake rate by roots of soybean plants was stimulated by the addition of K-malate to the external solution. It was decreased when phloem translocation was interrupted by hypocotyl girdling, and partially restored by malate addition to the medium, whereas glucose was ineffective. Introduction of K-malate into the transpiration stream using a split root system resulted in an enrichment of the phloem sap translocated back to the roots. This treatment resulted in an increase in both NO₃⁻ uptake and C excretion rates by roots. These results suggest that NO₃⁻ uptake by roots is dependent on the availability of shoot-borne, phloem-translocated malate. Shoot-to-root transport of malate stimulated NO₃⁻ uptake, and excretion of HCO₃⁻ ions was probably released by malate decarboxylation. NO₃⁻ uptake rate increased when the supply of NO₃⁻ to the shoot was increased, and decreased when the activity of nitrate reductase in the shoot was inhibited by WO₄²⁻. We conclude that in situ, NO₃⁻ reduction rate in the shoot may control NO₃⁻ uptake rate in the roots via the translocation rate of malate in the phloem.     

Comparison between NO(x) Evolution Mechanisms of Wild-Type and nr(1) Mutant Soybean Leaves

The nr₁ soybean (Glycine max [L.] Merr.) mutant does not contain the two constitutive nitrate reductases, one of which is responsible for enzymic conversion of nitrite to NO_x (NO + NO₂). It was tested for possible nonenzymic NO_x formation and evolution because of known chemical reactions between NO₂⁻ and plant metabolites and the instability of nitrous acid. It did not evolve NO_x during the in vivo NR assay, but intact leaves did evolve small amounts of NO_x under dark, anaerobic conditions. Experiments were conducted to compare NO₃⁻ reduction, NO₂⁻ accumulation, and the NO_x evolution processes of the wild type (cv Williams) and the nr₁ mutant. In vivo NR assays showed that wild-type leaves had three times more NO₃⁻ reducing capacity than the nr₁ mutant. NO_x evolution from intact, anerobic nr₁ leaves was approximately 10 to 20% that from wild-type leaves. Nitrite content of the nr₁ mutant leaves was usually higher than wild type due to low NO_x evolution. Lag times and threshold NO₂⁻ concentrations for NO_x evolution were similar for the two genotypes. While only 1 to 2% of NO_x from wild type is NO₂, the nr₁ mutant evolved 15 to 30% NO₂. The kinetic patterns of NO_x evolution with time weré completely different for the mutant and wild type. Comparisons of light and heat treatments also gave very different results. It is generally accepted that the NO_x evolution by wild type is primarily an enzymic conversion of NO₂⁻ to NO. However, this report concludes that NO_x evolution by the nr₁ mutant was due to nonenzymic, chemical reactions between plant metabolites and accumulated NO₂⁻ and/or decomposition of nitrous acid. Nonenzymic NO_x evolution probably also occurs in wild type to a degree but could be easily masked by high rates of the enzymic process.     

Sub-Parts-Per-Billion Nitrate Method: Use of an N(2)O-Producing Denitrifier to Convert NO(3) or NO(3) to N(2)O

A more sensitive analytical method for NO(3) was developed based on the conversion of NO(3) to N(2)O by a denitrifier that could not reduce N(2)O further. The improved detectability resulted from the high sensitivity of the Ni electron capture gas chromatographic detector for N(2)O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO(3) to N(2)O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 mug/liter) of NO(3) N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO(3) concentrations of <2 ppb of NO(3) N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of N in NO(3). This method avoids the incomplete reduction and contamination of the NO(3) -N by the NH(4) and N(2) pools which can occur by the conventional method of NO(3) analysis. N(2)O-producing denitrifier strains were also used to measure the apparent K(m) values for NO(3) use by these organisms. Analysis of N(2)O production by use of a progress curve yielded K(m) values of 1.7 and 1.8 muM NO(3) for the two denitrifier strains studied.     

Photosynthetic Assimilation of NO(3) by Intact Cells of the Cyanobacterium Anacystis nidulans: Influence of NO(3) and NH(4) Assimilation on CO(2) Fixation

Illuminated suspensions of Anacystis nidulans, supplied with saturating concentrations of CO₂ evolved O₂ at a greater rate when nitrate was simultaneously present. The extent of the stimulation of noncyclic electron flow induced by nitrate was dependent on light intensity, being maximal under light saturating conditions. Accordingly, nitrate depressed the rate of CO₂ fixation at limiting but not at saturating light, this depression reflecting the competition between both processes for assimilatory power. In contrast, ammonium stimulated CO₂ fixation at any light intensity assayed, the stimulation being dependent on the incorporation of ammonium to carbon skeletons. The positive effect of ammonium on CO₂ fixation also appeared to occur when nitrate was the nitrogen source, since with either nitrogen source an increase in the incorporation of newly fixed carbon into acid-soluble metabolites took place. From these results, the in vivo partitioning of assimilatory power between photosynthetic nitrogen and carbon assimilation and the quantitative and qualitative effects of inorganic nitrogen assimilation on CO₂ fixation are discussed.     

Relative Content of NO(3) and Reduced N in Xylem Exudate as an Indicator of Root Reduction of Concurrently Absorbed NO(3)

It is unclear if the relative content of NO₃⁻ and reduced N in xylem exudate provides an accurate estimate of the percentage reduction of concurrently absorbed NO₃⁻ in the root. Experiments were conducted to determine whether NO₃⁻ and reduced N in xylem exudate of vegetative, nonnodulated soybean plants (Glycine max [L.] Merr., `Ransom') originated from exogenous recently absorbed ¹⁵NO₃⁻ or from endogenous ¹⁴N pools. Plants either were decapitated and exposed to ¹⁵NO₃⁻ solutions for 2 hours or were decapitated for the final 20 minutes of a 50-minute exposure to ¹⁵NO₃⁻ in the dark and in the light. Considerable amounts of ¹⁴NO₃⁻ and reduced ¹⁴N were transported into the xylem, but almost all of the ¹⁵N was present as ¹⁵NO₃⁻. Dissimilar changes in transport of ¹⁴NO₃⁻, reduced ¹⁴N and ¹⁵NO₃⁻ during the 2 hours of sap collection resulted in large variability over time in the percentage of total N in the exudate which was reduced N. Over a 20-minute period the rate of ¹⁵N transport into the xylem of decapitated plants was only 21 to 36% of the ¹⁵N delivered to the shoot of intact plants. Based on the proportion of total ¹⁵N which was found as reduced ¹⁵N in exudate and in intact plants in the dark, it was estimated that 5 to 17% of concurrently absorbed ¹⁵NO₃⁻ was reduced in the root. This was much less than the 38 to 59% which would have been predicted from the relative content of total NO₃⁻ and total reduced N in the xylem exudate.     

Determination of N Abundance in Nanogram Pools of NO(3) and NO(2) by Denitrification Bioassay and Mass Spectrometry

Suspensions of two strains of Pseudomonas aeruginosa (ON12 and ON12-1) were used to reduce NO(3) and NO(2), respectively, to N(2)O. The evolved N(2)O was quantified by gas chromatography with electron capture detection, and the N abundance was determined by mass spectrometry with a special inlet system and triple-collector detection. Sample gas containing unknown N(2)O pools as small as 0.5 ng of N was analyzed by use of a spike technique, in which a reference gas of N(2)O of natural N abundance was added to obtain enough total N for the mass spectrometer. In NO(3) or NO(2) pools, the N abundance could be determined in samples as small as approximately 3.5 ng of N. No cross-contamination took place between the NO(3) and NO(2) pools. The excellent separation of NO(3) and NO(2) pools, small sample size required, and low contamination risk during N(2)O analysis offer great advantages in isotope studies of inorganic N transformations by, e.g., nitrifying or denitrifying bacteria in the environment.     

Exogenous NO(3) Influx and Endogenous NO(3) Efflux by Two Maize (Zea mays L.) Inbreds during Nitrogen Deprivation

The influence of nitrogen stress on net nitrate uptake resulting from concomitant ¹⁵NO₃⁻ influx and ¹⁴NO₃⁻ efflux was examined in two 12-day-old inbred lines of maize. Plants grown on ¹⁴NO₃⁻ were deprived of nitrogen for up to 72 hours prior to the 12th day and then exposed for 0.5 hour to 0.15 millimolar nitrate containing 98.7 atom% ¹⁵N. The nitrate concentration of the roots declined from approximately 100 to 5 micromolar per gram fresh weight during deprivation, and ¹⁴NO₃⁻ efflux was linearly related to root nitrate concentration. Influx of ¹⁵NO₃⁻ was suppressed in nitrogen-replete plants and increased with nitrogen deprivation up to 24 hours, indicating a dissipation of factors suppressing influx. Longer periods of nitrogen-deprivation resulted in a decline in ¹⁵NO₃⁻ influx from its maximal rate. The two inbreds differed significantly in the onset and extent of this decline, although their patterns during initial release from influx suppression were similar. Except for plants of high endogenous nitrogen status, net nitrate uptake was largely attributable to influx, and genetic variation in the regulation of this process is implied.     

Assimilation and Transport of Nitrogen in Nonnodulated (NO(3)-grown) Lupinus albus L

The response of nonnodulated white lupin (Lupinus albus L. cv. Ultra) plants to a range of NO₃ levels in the rooting medium was studied by in vitro assays of extracts of plant parts for NO₃ reductase (EC 1.6.6.1) activity, measurements of NO₃-N in plant organs, and solute analyses of root bleeding (xylem) sap and phloem sap from stems and petioles. Plants were grown for 65 days with 5 millimolar NO₃ followed by 10 days with 1, 5, 15, or 30 millimolar NO₃. NO₃ reductase was substrate-induced in all tissues. Roots contained 76, 68, 62 and 31% of the total NO₃ reductase activity of plants fed with 1, 5, 15, and 30 millimolar NO₃, respectively. Stem, petioles, and leaflets contained virtually all of the NO₃ reductase activity of a shoot, the activity in extracts of fruits amounting to less than 0.3% of the total enzyme recovered from the plant. Xylem sap from NO₃-grown nonnodulated plants contained the same organic solutes as from nodulated plants grown in the absence of combined N. Asparagine accounted for 50 to 70% and glutamine 10 to 20% of the xylem-borne N. The level of NO₃ in xylem sap amounted to 4, 13, 12, and 17% of the total xylem N at 1, 5, 15, and 30 millimolar NO₃, respectively. Xylem to phloem transfer of N appeared to be quantitatively important in supplying fruits and vegetative apices with reduced N, especially at low levels of applied NO₃. NO₃ failed to transfer in any quantity from xylem to phloem, representing less than 0.3% of the phloem-borne N at all levels of applied NO₃. Shoot organs were ineffective in storing NO₃. Even when NO₃ was supplied in great excess (30 millimolar level) it accounted for only 8% of the total N of stem and petioles, and only 2 and 1% of the N of leaflets and fruits, respectively.     

Economy of Carbon and Nitrogen in a Nodulated and Nonnodulated (NO(3)-grown) Legume

Partitioning and utilization of assimilated C and N were compared in nonnodulated, NO₃-fed and nodulated, N₂-fed plants of white lupin (Lupinus albus L.). The NO₃ regime used (5 millimolar NO₃) promoted closely similar rates of growth and N assimilation as in the symbiotic plants. Over 90% of the N absorbed by the NO₃-fed plants was judged to be reduced in roots. Empirically based models of C and N flow demonstrated that patterns of incorporation of C and N into dry matter and exchange of C and N among plant parts were essentially similar in the two forms of nutrition. NO₃-fed and N₂-fed plants transported similar types and proportions of organic solutes in xylem and phloem. Withdrawal of NO₃ supply from NO₃-fed plants led to substantial changes in assimilate partitioning, particularly in increased translocation of N from shoot to root. Nodulated plants showed a lower (57%) conversion of C or net photosynthate to dry matter than did NO₃-fed plants (69%), and their stems were only half as effective as those of NO₃-fed plants in xylem to phloem transfer of N supplied from the root. Below-ground parts of symbiotic plants consumed a larger share (58%) of the plants' net photosynthate than did NO₃-fed roots (50%), thus reflecting a higher CO₂ loss per unit of N assimilated (10.2 milligrams C/milligram N) by the nodulated root than by the root of the NO₃-fed plant (8.1 milligrams C/milligram N). Theoretical considerations indicated that the greater CO₂ output of the nodulated root involved a slightly greater expenditure for N₂ than for NO₃ assimilation, a small extra cost due to growth and maintenance of nodule tissue, and a considerably greater nonassimilatory component of respiration in root tissue of the symbiotic plant than in the root of the NO₃-fed plant.     

A search for interstellar oxiranecarbonitrile (C3H3NO)

We report a search in cold, quiescent and in hot core type interstellar molecular clouds for the small cyclic molecule oxiranecarbonitrile (C₃H₃NO), which has been suggested as a precursor of important prebiotic molecules. We have determined upper limits to the column density and fractional abundance for the observed sources and find that, typically, the fractional abundance by number relative to molecular hydrogen of C₃H₃NO is less than a few times 10^–10. This limit is one to two orders of magnitude less than the measured abundance of such similarly complex species as CH₃CH₂CN and HCOOCH₃ in well-studied hot cores. A number of astrochemical discoveries were made, including the first detection of the species CH₃CH₂CN in the massive star-forming clouds G34.3+0.2 and W51M and the first astronomical detections of some eight rotational transitions of CH₃CH₂CN, CH₃CCH, and HCOOCH₃. In addition, we found 8 emission lines in the 89 GHz region and 18 in the 102 GHz region which we were unable to assign.     

Charge equilibria and pK(a) of malvidin-3-glucoside by electrophoresis

Paper electrophoresis has been used over the pH range 1.2 to 10.4 to measure apparent pK(a) values for malvidin-3-O-glucoside of pK(a(1)) 1.76+/-0.07, pK(a(2)) 5.36+/-0.04, and pK(a(3)) 8.39+/-0.07. Using solvent partitioning between buffered aqueous solutions and n-octanol, several micro-pK(a) constants for malvidin-3-O-glucoside were also identified, highlighting the complex nature of malvidin-3-glucoside equilibria. As a nonspectrophotometric procedure, the charge-dependent electrophoretic mobility method provided independent information on the net charge and color of anthocyanin species at wine pH (ca. 3.6). At this pH, the color of malvidin-3-glucoside in red wines is consistent only with the uncharged quinonoidal base as a major colored component of the equilibria.     

Nucleotide Pools in Soybean Nodule Tissues, a Survey of NAD(P)/NAD(P)H Ratios and Energy Charge

The NAD⁺/NADH ratio was 12 in whole soybean nodules tissue,but only 2 in bacteroids, as a result of the high concentrationof NADH. By contrast, NADP⁺/NADPH ratios were less than unityin both nodules and bacteroids, being 0.28 and 0.37, respectively. The adenylate energy charge values in bacteroids and nodules,0.37 and 0.39, respectively, were remarkably low, and were insharp contrast to the normal value of 0.83 in root tissue. (Received July 19, 1988; Accepted March 9, 1989)     

Nitrogen Utilization in Lemna: II. Studies of Nitrate Uptake Using NO(3)

¹³N-labeled nitrate was used to trace short-term nitrate influx into Lemna gibba L. G3 in experiments where disappearance of both radioactivity and total nitrate from the incubation medium was measured continuously and simultaneously. In plants performing net nitrate uptake from an initial nitrate concentration of 40 to 60 micromolar, there was no discrepancy between net uptake and influx, irrespective of the N status of the plants, indicating that concomitant nitrate efflux was low or nil. Plants treated with tungstate to inactivate nitrate reductase were able to take up nitrate following induction of the uptake system by exposure to a low amount of nitrate. Also, in this case, net uptake was equivalent to influx. In tungstate-treated plants preloaded with nitrate, both net uptake and influx were nil. In contrast to these observations, a clear discrepancy between net uptake and influx was observed when the plants were incubated at an initial nitrate concentration of approximately 5 micromolar, where net uptake is low and eventually ceases. It is concluded that plasmalemma nitrate transport is essentially unidirectional in plants performing net uptake at a concentration of 40 to 60 micromolar, and that transport is nil when internal nitrate sinks (vacuole, metabolism) are eliminated. The efflux component becomes increasingly important when the external concentration approaches the threshold value for net nitrate uptake (the nitrate compensation point) where considerable exchange between internal and external nitrate occurs.     

Sodium-Stimulated NO(3) Uptake in Amaranthus tricolor L. Plants

Nitrate uptake of Na⁺ -deficient Amaranthus tricolor L. cv Tricolor seedlings from complete culture solution was stimulated by about 210% within 5 hours by application of 0.5 millimolar NaCl. From a Na⁺ -preloading experiment, intracellular Na⁺ was shown to be responsible for the stimulation of NO₃⁻ uptake. The results suggest a possible role of Na⁺ in NO₃⁻ uptake in C₄ plants.     

Uptake and Assimilation of NO(3) and NH(4) by Nitrogen-Deficient Perennial Ryegrass Turf

Assimilation of NO₃⁻ and NH₄⁺ by perennial ryegrass (Lolium perenne L.) turf, previously deprived of N for 7 days, was examined. Nitrogen uptake rate was increased up to four- to five-fold for both forms of N by N-deprivation as compared to N-sufficient controls, with the deficiency-enhanced N absorption persisting through a 48 hour uptake period. Nitrate, but not NH₄⁺, accumulated in the roots and to a lesser degree in shoots. By 48 hours, 53% of the absorbed NO₃⁻ had been reduced, whereas 97% of the NH₄⁺ had been assimilated. During the early stages (0 to 8 hours) of NO₃⁻ uptake by N-deficient turf, reduction occurred primarily in the roots. Between 8 and 16 hours, however, the site of reduction shifted to the shoots. Nitrogen form did not affect partitioning of the absorbed N between roots (40%) and shoots (60%) but did affect growth. Compared to NO₃⁻, NH₄⁺ uptake inhibited root, but not shoot, growth. Total soluble carbohydrates decreased in both roots and shoots during the uptake period, principally the result of fructan metabolism. Ammonium uptake resulted in greater total depletion of soluble carbohydrates in the root compared to NO₃⁻ uptake. The data indicate that N assimilation by ryegrass turf utilizes stored sugars but is also dependent on current photosynthate.     

Nitric oxide (NO) and NO cycle in myocardium: molecular, biochemical and physiological aspects

The article continues the series of our publications on the problem of nitric oxide (NO) and its cyclic conversion in mammals. This review is held to analysis of nitric oxide role in regulation of cardiovascular system and in alocation of NO-synthases in myocardium. Molecular, biochemical and cytophysiological aspects that linked, with spatial localization of NO-synthases and mechanisms of NO content regulation in myocardium are considered. The results of author's investigations along the cyclic convertion of NO and literature data about compartmentalization of NO-synthases in myocardium are included in this paper. The contradictory and dissimilar facts about regulatory and toxic role of nitric oxide in cardiovascular system are represented.     

Direct Association of Heat Shock Protein 20 (HSPB6) with Phosphoinositide 3-kinase (PI3K) in Human Hepatocellular Carcinoma: Regulation of the PI3K Activity

HSP20 (HSPB6), one of small heat shock proteins (HSPs), is constitutively expressed in various tissues and has several functions. We previously reported that the expression levels of HSP20 in human hepatocellular carcinoma (HCC) cells inversely correlated with the progression of HCC, and that HSP20 suppresses the growth of HCC cells via the AKT and mitogen-activated protein kinase signaling pathways. However, the exact mechanism underlying the effect of HSP20 on the regulation of these signaling pathways remains to be elucidated. To clarify the details of this effect in HCC, we explored the direct targets of HSP20 in HCC using human HCC-derived HuH7 cells with HSP20 overexpression. HSP20 proteins in the HuH7 cells were coimmunoprecipitated with the p85 regulatory subunit and p110 catalytic subunit of phosphoinositide 3-kinase (PI3K), an upstream kinase of AKT. Although HSP20 overexpression in HCC cells failed to affect the expression levels of PI3K, the activity of PI3K in the unstimulated cells and even in the transforming growth factor-α stimulated cells were downregulated by HSP20 overexpression. The association of HSP20 with PI3K was also observed in human HCC tissues in vivo. These findings strongly suggest that HSP20 directly associates with PI3K and suppresses its activity in HCC, resulting in the inhibition of the AKT pathway, and subsequently decreasing the growth of HCC.