Protein Tyrosine Phosphatases Mediate the Signaling Pathway of Stomatal Closure of Vicia faba L.

The regulation of stomatal movement is one of the most important signaling networks in plants.The H^ -ATPase at the plasma membrane of guard cells plays a critical role in the stomata opening, while there are some conflicting results regarding the effectiveness of the plasma membrane H^ -ATPase inhibitor,vanadate, in inhibiting stomata opening. We observed that 2 mmol/L vanadate hardly inhibited light-stimulated stomata opening in epidermal peels of Vicia faba L., but significantly inhibited dark- and ABA-induced stomatal closure. These results cannot be explained with the previous findings that H~-ATPase was inhibited by vanadate. In view of the fact that vanadate is an inhibitor of protein tyrosine phosphatases (PTPases),we investigated whether the stomatal movement regulated by vanadate is through the regulation of PTPase,As expected, phenylarsine oxide (PAO), a specific inhibitor of PTPase, has very similar effects and even more effective than vanadate. Typical PTPase activity was found in guard cells of V.faba; moreover, the phosphatase activity could be inhibited by both vanadate and PAO. These results not only provide a novel explanation for conflicting results about vanadate modulating stomatal movement, but also provide further evidence for the involvement of PTPases in modulating signal transduction of stomatal movement.     

Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α(TNF-α) conjugate for therapeutic application

To design a releasable PEGylated TNF-α(rPEG-TNF-α ), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF- (PEG-TNF- ), facilitating its clinical use for anti-tumor therapy. Comparative pharmaco- kinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ~60-fold greater than that of unmodified TNF-α . In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9- fold more potent, respectively, than PEG-TNF-α . Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α .     

Stratification of alkaline phosphatase in sediments of two urban lakes and its effect on phosphorus cycle

Phosphorus loadings in sediments play an important role in lake eutrophication and the progress of its recovery. The phosphorus release is controlled by physical, chemical and biological mechanisms. Alkaline phosphatase catalyzes remineralization of organic phosphorus and then it may be an important factor accelerating phosphorus cycling in sediments. In this paper, distributions, properties and function of alkaline phosphatase with depths in sediments of two urban lakes were discussed. Alkaline phosphatase activity (APA) in the sediments of Lake Yuehu decreased with the sediment depth. APA in sediments of Lake Yuehu was, mostly, inhibited by Phe and L-Cys; and inhibiting ability of Phe could be stronger than L-Cys. APA in deeper layer (20–30 cm) of sediments was more sensitive to the inhibitors than other layers, but range of variation in APA was most wide in the subsurface layer (10–20 cm). All the facts implied that alkaline phosphatase occurred in various forms (isoenzymes). APA in the sediments with different depths of Lake Donghu responded Phe differently. Reacted with Phe and incubated for 1 day, the amounts of SRP released by these sediments varied correspondingly. SRP on the overlying water in deeper layers (5–10 cm and 15–20 cm) of Site T1 was higher than that in surface layer (0–5 cm) of the same site, 1 day after incubation. Hence, the SRP release resulted, at least partially, from the hydrolysis of some liable organic phosphorus mediated by APA. Alkaline phosphatase in lake sediments plays an important role in the release of internal phosphorus loadings and eutrophication. A possible explanation for the sensitivity at deeper layers could be another active region of hydrolysis by alkaline phosphatase from organic phosphorus, which added a new dimension in phosphorus cycling mediated by some biochemical mechanisms.     

Effect of EGF on initiation of primordial follicle growth in ovary of newborn rat

The present study was designed to look at the effect of epidermal growth factor (EGF) and tomcie-stimulating hormone (FSH) on initiation of primordial follicle growth and differentiation in the ovary of newborn rat with a sensitive marker of proliferating cell nuclear antigen (PCNA). The results showed that more cuboidal granulosa cells (GC) were found in the ovary two days after injection of EGF. More proliferative GC were observed on D4. No such action of FSH on primordial follicles was demonstrated. Using in situ hybridization, inhibin a mRNA expression in GC was detected from D5, while FSH receptor (FSHR) mRNA expression started from D6 after birth. Both mRNAs increased following further development of the follicles. These results suggest that it is EGF, but not FSH, that may play a certain role in initiation of primordial follicle growth. FSH may be involved in further differentiation and growth of the early developmental follicles.     

Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

The temperature-dependence of infraeeliular free caleimn (Ca) was investigated in mdo-1 loaded ventricular myocytes from the ral, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-l at different temperatures was calibrated both al pll-tat and at @-stat . and the result demonstrated that the @-stat ralibration should be prettrred . Analysis of the fluoreseent image showed a striking increase of Ca2 as well as spontaneous caleiuni waves in ral cells, indicating an overloaded cakuum. In contrast, cardiac myocytes of the ground sqnirraf were found to keep a constant (Ca2 ) without caleium overload regardless of temperature variation. It is be-lieved that understanding of the mechanisms underlying the interccllular caleima homeostasis of hibrernators may lead to solutions of some medical questions .     

Characteristics of Triose Phosphate／Phosphate Translocator from Wheat and Its Role in the Distribution of Assimilates

In plants, triose phosphate/phosphate translocator (TPT) is the first regulation point forpartitioning of photosynthate between source and sink. Studies on the characteristic of TPT and itsregulation on the distribution of assimilates are critical for improving the utilization rate of photosyntheticassimilates. Chloroplasts with intactness of more than 91% and high purity were isolated from wheat( Triticurn aestivurn L. cv. Jing 411) leaves. Analysis of SDS-PAGE and labeling with an irreversible specificinhibitor, [H3]2^-DIDS (4, 4‘-diisothiocyano-2, 2‘-stilbenedisulfonate, DIDS) demonstrated that wheat TPTwas a chloroplast membrane protein with a 35 kD molecular weight, which comprised about 15% of the totalmembrane proteins of chloroplasts. Western blotting analysis showed that wheat TPT is uniquelydistributed in the envelope membrane of chloroplasts, but not detected in the membranes of vacuoles andmitochondria. The silicone-oil-layer centrifugation system was employed to study the kinetic properties ofTPT. The results showed that the maximal transport activity of TPT was the highest for dihydroxyacetonephosphate (DHAP)/inorganic phosphate (Pi), then for phosphoenolpyruvate (PEP)/Pi and glucose-6-phosphate (G6P)/Pi. The Km value of TPT was the lowest for DHAP, followed by Pi, PEP and G6P,therefore the most preferred substrate of TPT is DHAP. The transport of wheat TPT to DHAP was stronglyinhibited by DIDS with a degree of 95%. Inhibition of TPT transport activity led to an obvious accumulationof starch in chloroplasts, therefore the TPT protein of wheat controls the export of TP out of chloroplastsinto cytosol. Except for the need of participating in the Calvin cycle, the ratio of TP exported out ofchloroplast to the one used for synthesizing starch was at least 93.6:6.4. The TPT protein from wheat hasmuch high transport efficiency, which plays an important role in the regulation of the distribution ofassimilates in wheat chloroplasts.     

Histochemical and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog submandibular glands.

p-Nitrophenyl phosphatase (p-NPPase) activity of (Na+-K+)-activated adenosine triphosphatase ((Na+-K+)-ATPase) on the acinar cells of dog submandibular gland was demonstrated by using light microscopy. The reaction products of p-NPPase of fresh frozen sections were seen to be localized on the basal parts of acini, and disappeared when the sections were incubated in medium containing 10(-3) Mouabain or in a K-free medium. Under the electron microscope, the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells. On the microvilli of the luminal plasma membrane of the acinar cell, a small quantity of the reaction products was also present. This localization of ATPase reaction products on the serous and mucous cells seemed to coincide well with that of p-NPPase activity observed on the acini under light microscopy. Possible explanations are given regarding distribution of the above mentioned enzymes in relation to the cation transport of the plasma membrane. Structural and functional asymmetrical properties of acinar cells of the dog submandibular gland are also discussed.     

Activation of ouabain-sensitive p-nitrophenylphosphatase by carbachol and cGMP in rat submandibular gland

Na+,K+-ATPase activity was monitored by measuring ouabain-sensitive K+-dependent p-nitrophenylphosphatase (p-NPPase) activity in rat submandibular gland slices. Carbachol (carbamylcholine chloride) stimulated the p-NPPase activity in the presence of calcium but not in its absence. Carbachol activation of the enzyme was totally ouabain sensitive and could be blocked by atropine. A minimal requirement of sodium ion extracellularly was required for this carbachol stimulation. cGMP and its dibutyryl analogue was also effective in stimulating the enzyme activity, whereas, cAMP was ineffective. Calcium, however, was not required for cGMP activation of the p-NPPase activity. The result indicates that calcium is the second messenger and cGMP is the tertiary connection between cholinergic stimulation and Na+,K+-ATPase activation in these glands. Activation of Na+,K+-ATPase is postulated to be responsible for primary fluid formation.     

Isolation of basolateral and brush-border membranes from the rabbit kidney cortex. Vesicle integrity and membrane sidedness of the basolateral fraction

A rapid and reproducible method has been developed for the simultaneous isolation of basolateral and brush-border membranes from the rabbit renal cortex. The basolateral membrane preparation was enriched 25-fold in (Na+ + K+)-ATPase and the brush-border membrane fraction was enriched 12-fold in alkaline phosphatase, whereas the amount of cross-contamination was low. Contamination of these preparations by mitochondria and lysosomes was minimal as indicated by the low specific activities of enzyme markers, i.e., succinate dehydrogenase and acid phosphatase. The basolateral fraction consisted of 35-50% sealed vesicles, as demonstrated by detergent (sodium dodecyl sulfate) activation of (Na+ + K+)-ATPase activity and [3H]ouabain binding. The sidedness of the basolateral membranes was estimated from the latency of ouabain-sensitive (Na+ + K+)-ATPase activity assayed in the presence of gramicidin, which renders the vesicles permeable to Na+ and K+. These studies suggest that nearly 90% of the vesicles are in a right-side-out orientation.     

Effects of palytoxin on cation occlusion and phosphorylation of the (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase

Palytoxin (PTX) inhibits the (Na(+) + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from P(i); and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+, K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from P(i) insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+, K(+)-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside "gate" of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.     

Studies on vinblastine-induced autophagocytosis in mouse liver. II. Origin of membranes and acquisition of acid phosphatase

The origin of the membranes of autophagic vacuoles (AV) and acquisition of acid phosphatase into AV's were studied in vinblastine-induced autophagocytosis (VBL, 50 mg/kg, i.p.) in mouse hepatocytes. Using unbuffered OsO4, very intense staining was observed in the outer cisternae of the Golgi apparatus and also frequently in the cavity between the double membranes obviously destined to form AV's as well as in the cavity between the double membranes of newly formed AV's. There may occur a transformation process in the membranes limiting an AV analogous to that observed at the Golgi cisternae. The transformation of the outer AV membrane occurs independently of fusion with lysosomes. Inosine diphosphatase activity was localized within the cisternae and on the membranes of the endoplasmic recticulum and occasionally within the innermost cisterna of the Golgi apparatus. The results together with the unbuffered OsO4-staining pattern suggest that the membranes of most AV's are derived from the transformed smooth surfaced cisternae of the endoplasmic reticulum which do not have inosine diphosphatase activity. Acid phosphatase activity was localized in lysosomes, occasionally within the innermost cisternae of the Golgi apparatus, between the double membranes of a few newly formed AV's and within most older single membranes of a few newly formed AV's and within most older single membrane-limited AV's. VBL did not prevent the fusion of lysosomes with AV's.     

Time course of neutral red-induced autophagy in murine pancreatic acinar cells. A morphometrical and cytochemical study.

The knowledge of the time course of the influences of chemicals on autophagy is of great importance in the study of their modes of action and hence provides information relating the mechanism and dynamics of this catabolic process. Neutral red (NR) treatment has long been used to produce an accumulation of autolysosomes in different cell types. In the present study early (AV1), advanced (AV2) and late (AV3), as well as complex (fused) AVs (AVc) were distinguished. In our morphometrical measurements, we found all these AV subcompartments significantly expanded as early as 30 min after the injection of NR (0.4 mg/g b.wt.), i.e. a large number of AVs accumulated in the cells. Since cytoplasmic volume fraction (CVF) of AV increased 3-fold during this early period we conclude that, unlike vinblastine, NR is not a fusion inhibitor. Accumulation of AV1 (3-fold) in the presence of fusions possibly indicates that NR stimulates formation of AVs in this early period, after the accumulation of AVs continued. The maximal CVF of AVs were measured at 4 h, when 7.6% of the cytoplasmic volume was sequestered into the AV compartment, two third of which came from AV3. This finding indicates that NR is probably an inhibitor of intravacuolar degradation. However, the high rate of accumulation of AV2, AV3, and total AVs including a slower but still pronounced accumulation of AV1 cannot be explained solely from inhibition of degradation, but indicates a stimulated segregation (AV formation). Our results therefore argue for a possible coupling of the regulation of autophagic segregation and degradation since vinblastine and possibly some other degradation inhibitors were also found to stimulate AV formation in other studies. Another goal of this study was to follow the time course of changes in distribution of certain lysosomal enzymes after NR treatment. According to our enzyme cytochemical studies, acid phosphatase (AP) of untreated cells is mainly located in large and small lysosomal elements of the Golgi zone, aryl sulfatase B (AS) in trans-Golgi elements including pre-secretory granules and trimetaphosphatase (TP) in basal lysosomes. After NR injection TP seemed to appear first in AV1 whereas AP activity was characteristic of more advanced AVs. AS activity only occasionally appeared in AV3 and exclusively at late times after NR injection.     

Further evidence for the existence of an intrinsic bicarbonate-stimulated Mg2+-ATPase in brush border membranes isolated from rat kidney cortex

Summary The aim of this study was to provide further evidence for the existence of a nonmitochondrial bicarbonate-stimulated Mg²⁺-ATPase in brush border membranes derived from rat kidney cortex. A plasma membrane fraction rich in brush border microvilli and a mitochondrial fraction were isolated by differential centrifugation. Both fractions contain a Mg²⁺-ATPase activity which can be stimulated by bicarbonate. The two Mg²⁺-ATPases are stimulated likewise by chloride, bicarbonate, and sulfite or inhibited by oligomycin and aurovertin, though to different degrees. In contrast to these similarities, only the Mg²⁺-ATPase activity of the mitochondrial fraction is inhibited by atractyloside, a substance which blocks an adenine nucleotide translocator in the inner mitochondrial membrane. On the other hand, filipin, an antibiotic that complexes with cholesterol in the membranes inhibits exclusively the Mg²⁺-ATPase of the cholesterol-rich brush border membranes. Furthermore it could be demonstrated by the use of bromotetramisole, an inhibitor of alkaline phosphatase activity, that the Mg²⁺-ATPase activity in the membrane fraction is not due to the presence of the highly active alkaline phosphatase in these membranes. These results support the assumption that an intrinsic bicarbonate-stimulated Mg²⁺-ATPase is present in rat kidney brush border membranes.     

Proton ATPase in rat renal cortical endocytotic vesicles

To relate ATPase activity to the ATP-driven H+-pump in rat renal endocytotic vesicles we applied an in vitro coupled optical test and a Pi-liberation assay. Endocytotic vesicles contain an ouabain-, vanadate- and oligomycin-insensitive ATPase. The ionophores for K+ and H+, valinomycin and carbonylcyanide p-chloro-methoxyphenylhydrazone (CCCP), respectively, stimulated ATPase activity, indicating its relation to the electrogenic H+-pump. This conclusion is supported by a similar distribution on a Percoll gradient of ATP-driven H+ uptake into endosomes and ionophore-stimulated ATPase activity. Coupled optical and Pi-liberation assays were then used to characterize the H+-ATPase with respect to the requirement for pH, nucleotides, anions, and mono- and divalent cations. The H+-ATPase activity was decreased by widely used blockers: N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and diethylstilbestrol (DES). Different sensitivities to these blockers proved that alkaline phosphatase and H+-ATPase are separate entities. To investigate whether the NEM-, DCCD- and DES-sensitive ATPase activity is confined to intact endocytotic vesicles, cellular membranes from rat kidney cortex were separated on a Percoll density gradient. Surprisingly, endocytotic vesicles contain only a small fraction of the total NEM-, DCCD- and DES-sensitive ATPase activity. The majority of the blocker-sensitive ATPases belongs to membranes of as yet undefined cellular origin.     

Lysophosphatidylcholine specifically stimulates plasma membrane H(+)-ATPase from corn roots

The plasma membrane H(+)-ATPase activity from corn seedling roots is shown to be stimulated 3- to 4-fold by the addition of lysophosphatidylcholine (lysoPC). This effect clearly differs from that of other detergents by both the magnitude and the absence of inhibition at higher concentrations. LysoPC decreases the apparent Km for MgATP, increases Vmax of the ATPase reaction but does not change its pH optimum. On the contrary, the acid phosphatase activity associated with plasma membranes is not influenced by lysoPC. A lysoPC stimulation is also demonstrated for the solubilized preparation of the H(+)-ATPase. It is assumed that lysoPC stimulation of the plant plasma membrane H(+)-ATPase is not only due to permeabilization of the vesicles for MgATP, but also to direct action on the enzyme.     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the membranes of autophagic vacuoles (AV) and acquisition of acid phosphatase into AV's were studied in vinblastine-induced autophagocytosis (VBL, 50 mg/kg, i.p.) in mouse hepatocytes. Using unbuffered OsO₄, very intense staining was observed in the outer cisternae of the Golgi apparatus and also frequently in the cavity between the double membranes obviously destined to form AV's as well as in the cavity between the double membranes of newly formed AV's. There may occur a transformation process in the membranes limiting an AV analogous to that observed at the Golgi cisternae. The transformation of the outer AV membrane occurs independently of fusion with lysosomes. Inosine diphosphatase activity was localized within the cisternae and on the membranes of the endoplasmic reticulum and occasionally within the innermost cisterna of the Golgi apparatus. The results together with the unbuffered OsO₄-staining pattern suggest that the membranes of most AV's are derived from the transformed smooth surfaced cisternae of the endoplasmic reticulum which do not have inosine diphosphatase activity. Acid phosphatase activity was localized in lysosomes, occasionally within the innermost cisternae of the Golgi apparatus, between the double membranes of a few newly formed AV's and within most older single membrane-limited AV's. VBL did not prevent the fusion of lysosomes with AV's.This research was supported by grants from the Ellen and Artturi Nyyssönen Foundation and the Heikki and Hilma Honkanen Foundation