Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins

Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem, mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. `Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The identified xylem–preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one α-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing may yield information complementary to that from EST sequencing strategies. Received: 28 June 1999 / Accepted: 3 September 1999     

Biotechnology applications of amino acids in protein purification and formulations

Summary. Amino acids are widely used in biotechnology applications. Since amino acids are natural compounds, they can be safely used in pharmaceutical applications, e.g., as a solvent additive for protein purification and as an excipient for protein formulations. At high concentrations, certain amino acids are found to raise intra-cellular osmotic pressure and adjust to the high salt concentrations of the surrounding medium. They are called “compatible solutes”, since they do not affect macromolecular function. Not only are they needed to increase the osmotic pressure, they are known to increase the stability of the proteins. Sucrose, glycerol and certain amino acids were used to enhance the stability of unstable proteins after isolation from natural environments. The mechanism of the action of these protein-stabilizing amino acids is relatively well understood. On the contrary, arginine was accidentally discovered as a useful reagent for assisting in the refolding of recombinant proteins. This effect of arginine was ascribed to its ability to suppress aggregation of the proteins during refolding, thereby increasing refolding efficiency. By the same mechanism, arginine now finds much wider applications than previously anticipated in the research and development of proteins, in particular in pharmaceutical applications. For example, arginine solubilizes proteins from loose inclusion bodies, resulting in efficient production of active proteins. Arginine suppresses protein–protein interactions in solution and also non-specific adsorption to gel permeation chromatography columns. Arginine facilitates elution of bound proteins from various column resins, including Protein-A or dye affinity columns and hydrophobic interaction columns. This review covers various biotechnology applications of amino acids, in particular arginine.     

A MBP-FAAH fusion protein as a tool to produce human and rat fatty acid amide hydrolase: expression and pharmacological comparison

Summary. Fatty acid amide hydrolase (FAAH), a membrane-anchored enzyme responsible for the termination of endocannabinoid signalling, is an attractive target for treating conditions such as pain and anxiety. Inhibitors of the enzyme, optimized using rodent FAAH, are known but their pharmacology and medicinal chemistry properties on the human FAAH are missing. Therefore recombinant human enzyme would represent a powerful tool to evaluate new drug candidates. However, the production of high amounts of enzyme is hampered by the known refractiveness of FAAH to overexpression. Here, we report the successful overexpression of rat and human FAAH as a fusion to the E. coli maltose-binding protein, retaining catalytic properties of native FAAH. Several known FAAH inhibitors were tested and differences in their potencies toward the human and rat FAAH were found, underscoring the importance of using a human FAAH in the development of inhibitors. Authors’ address: Didier M. Lambert, Unité de Chimie pharmaceutique et de Radiopharmacie, Université catholique de Louvain, Avenue E. Mounier 73.40, 1200 Bruxelles, Belgique     

Ubiquitin dependent and independent protein degradation in the regulation of cellular polyamines

Summary. Protein degradation mediated by the ubiquitin/proteasome system is the major route for the degradation of cellular proteins. In this pathway the ubiquitination of the target proteins is manifested via the concerted action of several enzymes. The ubiquinated proteins are then recognized and degraded by the 26S proteasome. There are few reports of proteins degraded by the 26S protesome without ubiquitination, with ornithine decarboxylase being the most notable representative of this group. Interestingly, while the degradation of ODC is independent of ubiquitination, the degradation of other enzymes of the polyamine biosynthesis pathway is ubiquitin dependent. The present review describes the degradation of enzymes and regulators of the polyamine biosynthesis pathway.     

Catabolism of native and oxidized low density lipoproteins: in vivo insights from small animal positron emission tomography studies

Summary. The human organism is exposed to numerous processes that generate reactive oxygen species (ROS). ROS may directly or indirectly cause oxidative modification and damage of proteins. Protein oxidation is regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to Alzheimer’s disease and atherosclerosis. As a representative example, oxidation of low density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Data concerning the role of circulating oxidized LDL (oxLDL) in the development and outcome of diseases are scarce. One reason for this is the shortage of methods for direct assessment of the metabolic fate of circulating oxLDL in vivo. We present an improved methodology based on the radiolabelling of apoB-100 of native LDL (nLDL) and oxLDL, respectively, with the positron emitter fluorine-18 (¹⁸F) by conjugation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). Radiolabelling of both nLDL and oxLDL using [¹⁸F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively, in vitro. The method was further evaluated with respect to the radiopharmacological properties of both [¹⁸F]fluorobenzoylated nLDL and oxLDL by biodistribution studies in male Wistar rats. The metabolic fate of [¹⁸F]fluorobenzoylated nLDL and oxLDL in rats in vivo was further delineated by dynamic positron emission tomography (PET) using a dedicated small animal tomograph (spatial resolution of 2 mm). From this study we conclude that the use of [¹⁸F]FB-labelled LDL particles is an attractive alternative to, e.g., LDL iodination methods, and is of value to characterize and to discriminate the kinetics and the metabolic fate of nLDL and oxLDL in small animals in vivo.     

Purification and structural analysis of an abundant thaumatin-like protein from ripe banana fruit

 The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Characterization of the protein and molecular cloning of the corresponding gene from banana demonstrated that the native protein consists of a single polypeptide chain of 200 amino acid residues. Molecular modelling further revealed that the banana thaumatin-like protein (Ban-TLP) adopts an overall fold similar to that of thaumatin and thaumatin-like PR-5 proteins. Although the banana protein exhibits an electrostatically polarized surface, which is believed to be essential for the antifungal properties of TLPs, it is apparently devoid of antifungal activity towards pathogenic fungi. It exhibits a low but detectable in vitro endo-β-1,3-glucanase (EC 3.2.1.x) activity. As well as being present in fruits, Ban-TLP also occurs in root tips where its accumulation is enhanced by methyl jasmonate treatment of plants. Pulp of plantains (Musa acuminata) also contains a very similar TLP, which is even more abundant than its banana homologue. Our results demonstrate for the first time that fruit-specific (abundant) TLPs are not confined to dicots but occur also in fruits of monocot species. The possible role of the apparent widespread accumulation of fruit-specific TLPs is discussed. Received: 7 January 2000 / Accepted: 26 April 2000     

Fumaric acid: an overlooked form of fixed carbon in Arabidopsis and other plant species

Photoassimilates are used by plants for production of energy, as carbon skeletons and in transport of fixed carbon between different plant organs. Many studies have been devoted to characterizing the factors that regulate photoassimilate concentrations in different plant species. Most studies examining photoassimilate concentrations in C₃ plants have focused on analyzing starch and soluble sugars. However, work presented here demonstrates that a number of C₃ plants, including the popular model organism Arabidopsis thaliana (L.) Heynh., and agriculturally important plants, such as soybean, Glycine max (L.) Merr., contain significant quantities of fumaric acid. In fact, fumaric acid can accumulate to levels of several milligrams per gram fresh weight in Arabidopsis leaves, often exceeding those of starch and soluble sugars. Fumaric acid is a component of the tricarboxylic acid cycle and, like starch and soluble sugars, can be metabolized to yield energy and carbon skeletons for production of other compounds. Fumaric acid concentrations increase with plant age and light intensity in Arabidopsis leaves. Moreover, Arabidopsis phloem exudates contain significant quantities of fumaric acid, raising the possibility that fumaric acid may function in carbon transport. Received: 11 February 2000 / Accepted: 1 April 2000     

Kinetic and proteomic analyses of <Emphasis Type="Italic">S</Emphasis>-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism

Summary. Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in V_max with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented. Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.     

The role of growth hormone and amino acids on brain protein synthesis in aged rats given proteins of different quantity and quality

Summary. The purpose of the present study was to determine whether the regulation of brain protein synthesis was mediated through changes in the plasma concentrations of insulin and growth hormone (GH), and whether the concentrations of amino acids in the brain and plasma regulate the brain protein synthesis when the quantity and quality of dietary protein is manipulated. Two experiments were done on three groups of aged rats given diets containing 20% casein, 5% casein or 0% casein (Experiment 1), and 20% casein, 20% gluten, or 20% gelatin (Experiment 2) for 1 d (only one 5-h period) after all rats were fed the 20% casein diet for 10 d (only 5-h feeding per day). The aggregation of brain ribosomes, the concentration in plasma GH, and the branched chain amino acids in the plasma and cerebral cortex declined with a decrease of quantity and quality of dietary protein. The concentration of plasma insulin did not differ among groups. The results suggest that the ingestion of a higher quantity and quality of dietary protein increases the concentrations of GH and several amino acids in aged rats, and that the concentrations of GH and amino acids are at least partly related to the mechanism by which the dietary protein affects brain protein synthesis in aged rats.     

The genes ABI1 and ABI2 are involved in abscisic acid- and drought-inducible expression of the Daucus carota L. Dc3 promoter in guard cells of transgenic Arabidopsis thaliana (L.) Heynh.

 The ABA INSENSITIVE1 (ABI1) and ABI2 genes encode homologous type-2C protein phosphatases with redundant yet distinct functions in abscisic acid (ABA) responses. Results from Northern blot analysis showed that ABA- and mannitol-inducible expression of the COR47 and COR78/LTI78/RD29A (COR78) genes was more impaired in the abi2 mutant of Arabidopsis thaliana (L.) Heynh than in the abi1 mutant. Furthermore, ABA-plus-mannitol treatments were additive towards COR47 gene expression; however, the ABA-deficient aba1 mutant showed reduced COR expression relative to the wild type in response to mannitol and ABA-plus-mannitol treatments. These results support the notion that drought- and ABA-signalling pathways are separate yet overlapping. To facilitate quantitative analysis of the genetic control of tissue-specific ABA- and desiccation-response pathways, we analyzed ABA- and mannitol-inducible expression of a carrot (Daucus carota L.) Dc3 promoter:uidA (β-glucuronidase; GUS) chimaeric reporter (Dc3-GUS) in transgenic wild-type, ABA-deficient aba1, and ABA-insensitive abi1 and abi2 mutants. The Dc3 promoter directed ABA- and mannitol-inducible GUS expression in Arabidopsis guard cells and the two treatments were additive. The aba1, abi1, and abi2 mutant genotypes had reduced GUS expression in guard cells of cotyledons in response to mannitol, whereas abi1 and abi2 mutants were reduced in ABA-inducible GUS expression, consistent with overlapping ABA- and drought-response pathways. Quantitative fluorometric GUS assays of leaf extracts showed that abi2 mutants responded less to exogenous ABA than did abi1 mutants, and abi2 mutants responded more to mannitol than did abi1 mutants. We conclude that Dc3-GUSArabidopsis is a tractable system in which to study tissue-specific ABA and drought signalling and suggest that ABI2 functions predominantly over ABI1 in COR78 and COR47 gene expression and guard-cell Dc3-GUS expression. Received: 23 May 1999 / Accepted: 3 December 1999     

The long history of Cannabis and its cultivation by the Romans in central Italy, shown by pollen records from Lago Albano and Lago di Nemi

The cores from the Albano and Nemi lakes, near Rome, were studied within the European Union funded PALICLAS project and provided high resolution records of the Late-glacial and Holocene. Pollen evidence of increasing human influence on vegetation was recorded in the Holocene parts of both diagrams, and the Cannabis (hemp) curve was one of the major signs. In this paper we present unambiguous pollen evidence from the Cannabaceae records for the cultivation of hemp in central Italy by the Romans. The oldest records of Cannabis and Humulus (hop) date from to the Late-glacial. Hop pollen values rise during the mid Holocene, while hemp pollen becomes more abundant from ca. 3000 cal B.P. onwards. The highest earliest hemp peak (21%) is dated to the 1st century A.D. This ‘Cannabis phase’, with the abrupt rise of hemp pollen soon after the rise of cultivated trees (Castanea, Juglans and Olea) is associated with the increase in cereals and ruderal plants. This unambiguous proof of cultivation by Romans around 2000 B.P. occurs as well as a long lasting pre-Roman presence of hemp in the area, which is natural and possibly also anthropogenic. Subsequent clear episodes of cultivation in the medieval period were found. Received February 4, 2002 / Accepted September 13, 2002 Correspondence to: Anna Maria Mercuri, e-mail: mercuri.annamaria@unimo.it     

Prediction of protein homo-oligomer types by pseudo amino acid composition: Approached with an improved feature extraction and Naive Bayes Feature Fusion

Summary. The interaction of non-covalently bound monomeric protein subunits forms oligomers. The oligomeric proteins are superior to the monomers within the scope of functional evolution of biomacromolecules. Such complexes are involved in various biological processes, and play an important role. It is highly desirable to predict oligomer types automatically from their sequence. Here, based on the concept of pseudo amino acid composition, an improved feature extraction method of weighted auto-correlation function of amino acid residue index and Naive Bayes multi-feature fusion algorithm is proposed and applied to predict protein homo-oligomer types. We used the support vector machine (SVM) as base classifiers, in order to obtain better results. For example, the total accuracies of A, B, C, D and E sets based on this improved feature extraction method are 77.63, 77.16, 76.46, 76.70 and 75.06% respectively in the jackknife test, which are 6.39, 5.92, 5.22, 5.46 and 3.82% higher than that of G set based on conventional amino acid composition method with the same SVM. Comparing with Chou’s feature extraction method of incorporating quasi-sequence-order effect, our method can increase the total accuracy at a level of 3.51 to 1.01%. The total accuracy improves from 79.66 to 80.83% by using the Naive Bayes Feature Fusion algorithm. These results show: 1) The improved feature extraction method is effective and feasible, and the feature vectors based on this method may contain more protein quaternary structure information and appear to capture essential information about the composition and hydrophobicity of residues in the surface patches that buried in the interfaces of associated subunits; 2) Naive Bayes Feature Fusion algorithm and SVM can be referred as a powerful computational tool for predicting protein homo-oligomer types.     

Influence of nitric oxide synthase activity on amino acid concentration in the quinolinate lesioned rat striatum: A microdialysis and histochemical study

Summary. The influence of nitric oxide synthase (NOS) activity on the KCl-evoked amino acid concentrations was investigated by in vivo microdialysis in the striatum in a rat model of excitotoxic lesion. Basal microdialysate levels of amino acids decreased during the quinolinic acid-induced neurodegeneration process, except for glutamine that increased initially and returned to control values 30 days after quinolinic acid exposure. KCl-evoked increase of extracellular amino acid concentration was reduced due to NOS activity in the striatum of both controls and lesioned animals, except for 120 days after quinolinic acid injection. These changes of amino acid concentrations in microdialysates correlated with the known biochemistry of the consecutive domineered cell types during the lesion process as revealed by histochemistry for NOS, NADPH-diaphorase, GFAP and isolectin B4. The present data provide direct evidence that NOS activity can modulate extracellular amino acid concentrations in the striatum not only under physiological conditions, but also during a pharmacologically induced lesion process and, thus, suggests that nitric oxide affects neurodegeneration via this pathway. Received October 20, 1999; Accepted February 25, 2000     

The pattern of acropetal and basipetal cytoplasmic streaming velocities in Chara rhizoids and protonemata, and gravity effect on the pattern as measured by laser-Doppler-velocimetry

 The spatial pattern of acropetal and basipetal cytoplasmic streaming velocities has been studied by laser-Doppler-velocimetry (LDV) in the positively gravitropic (downward growing) rhizoids of Chara globularis Thuill. and for the first time in the negatively gravitropic (upward growing) protonemata. The LDV method proved to be precise and yielded reproducible results even when tiny differences in velocities were measured. In the apical parts of the streaming regions of both cell types, acropetal streaming was faster than basipetal streaming. Starting at the apical reversal point of streaming, the velocity increased basipetally with the distance from that point and became fairly constant close to the basal reversal point; subsequently, the velocity decreased slightly acropetally as the apical reversal point was again approached. There was no change in velocity at the basal reversal point. However, at the apical reversal point there was an abrupt decrease in velocity. The pattern of the ratio of acropetal to basipetal streaming velocity (VR) was a function of the relative distance of the site of measurement from the apical reversal point rather than a function of the absolute distance. Upon inversion of the rhizoids, the VR decreased on average by 3.8% (±0.4%), indicating that the effect of gravity on the streaming velocity was merely physical and without a physiological amplification. Rhizoids that had developed on the slowly rotating horizontal axis of a clinostat, and had never experienced a constant gravity vector, were similar to normally grown rhizoids with respect to VR pattern. In protonemata, the VR pattern was not significantly different from that in rhizoids although the direction of growth was inverse. In rhizoids, oryzalin caused the polar organization of the cell to disappear and nullified the differences in streaming velocities, and cytochalasin D decreased the velocity of basipetal streaming slightly more than that of acropetal streaming. Cyclopiazonic acid, known as an inhibitor of the Ca²⁺-ATPase of the endoplasmic reticulum, also reduced the streaming velocities in rhizoids, but had slightly more effect on the acropetal stream. It is possible that the endogenous difference in streaming velocities in both rhizoids and protonemata is caused by differences in the cytoskeletal organization of the opposing streams and/or loading of inhibitors (like Ca²⁺) from the apical/subapical zone into the basipetally streaming endoplasm. Received: 4 October 1999 / Accepted: 4 November 1999     

Control of gravimorphogenesis by auxin: accumulation pattern of CS-IAA1 mRNA in cucumber seedlings grown in space and on the ground

Cucumber (Cucumis sativus L.) seedlings grown in microgravity developed a peg on each side of the transition zone between hypocotyl and root, whereas seedlings grown in a horizontal position on the ground developed a peg on the concave side of the gravitropically bending transition zone. The morphological features of the space-grown seedlings were similar to those of seedlings grown in a vertical position on the ground with their radicles pointing down: both became two-pegged seedlings. Morphogenesis of cucumber seedlings is thus inhibited by gravity. Analysis by in-situ hybridization of an auxin-inducible gene, CS-IAA1, showed that its mRNA accumulated to a much greater extent on the lower side of the transition zone in the horizontally placed seedlings on the ground just prior to and during the initiation period of peg formation. On the other hand, when seedlings were grown in microgravity or in a vertical position on the ground, accumulation of CS-IAA1 mRNA occurred all around the transition zone. Accumulation of CS-IAA1 mRNA in horizontally grown seedlings appreciably decreased on the upper side of the transition zone and increased on the lower side upon gravistimulation, compared with the two-pegged seedlings. Application of IAA to seedlings in a horizontal position caused the development of a peg on each side of the transition zone, or a collar-like protuberance, depending on the concentration used. These results suggest that upon gravistimulation the auxin concentration on the upper side of the horizontally placed transition zone is reduced to a level below the threshold value necessary for peg formation. Space-grown seedlings of cucumber might develop two pegs symmetrically because the auxin level in the entire transition zone is maintained above the threshold. This spaceflight experiment verified for the first time that auxin does not redistribute in microgravity. Received: 10 February 2000 / Accepted: 15 March 2000     

The role of L-arginine in toxic liver failure: interrelation of arginase,polyamine catabolic enzymes and nitric oxide synthase

Summary. The existing interrelation in metabolic pathways of L-arginine to polyamines, nitric oxide (NO) and urea synthesis could be affected in sepsis, inflammation, intoxication and other conditions. The role of polyamines and NO in the toxic effect of mercury chloride on rat liver function was studied. Administration of mercury chloride for 24 h led to significantly elevated plasma activities of Alanine transaminase (ALT) and Aspartate transaminase (AST). Malondyaldehyde (MDA) levels were unaffected (p > 0.05) and arginase activity was significantly decreased (p < 0.05) while nitrate/nitrite production was significantly elevated (p < 0.001) in liver tissue. Polyamine oxidase (PAO) and diamine oxidase (DAO) activities, enzymes involved in catabolism of polyamines, were decreased. L-arginine supplementation to intoxicated rats potentiated the effect of mercury chloride on NO production and it was ineffective on arginase activity. Results obtained in this study show that mercury chloride-induced toxicity leads to abnormally high levels of ALT and AST that may indicate liver damage with the involvement of polyamine catabolic enzymes and NO.     

Immunochemical analysis of protein expression in breast epithelial cells transformed by estrogens and high linear energy transfer (LET) radiation

Breast cancer is a complex disease involving numerous genetic aberrations. Immunochemical analysis of protein expression is presented in a human breast epithelial cell line neoplastically transformed by high linear energy transfer (LET) α particle radiation in the presence of 17β estradiol (E) and in the parental human breast epithelial cell line (MCF-10F) which served as a non-tumorigenic control. The aim of this work was to determine the levels of mRNA and protein expression in control and transformed cells at various stages of the neoplastic process. The levels of mRNA and protein expression of PCNA, c-fos, JNK2 and Fra-1 were increased in the transformed cell line compared to the levels in non-tumorigenic control cells. The transforming factor Rho A was significantly increased only in the tumor cell line. Furthermore, the levels of mRNA and protein expression of ErbB2 were significantly increased in the transformed cell line and in tumor cells derived from the transformed cells after injecting them into nude mice. A decrease in RbA/p48 protein expression and mRNA levels was observed in cells treated with double doses of α particle radiation in the presence of estrogen, regardless of tumorigenicity. Such expression was lower than that in the control untreated MCF-10F cells. In summary, these studies show that estrogen and high LET-radiation induce changes in oncoprotein expression and mRNA levels of human breast cell lines. These changes are indicative of a cascade of events that characterize the process of cell transformation in breast cancer. These results provide evidence that multiple steps with consecutive changes are involved when normal cells become tumorigenic cells as a result of α particle irradiation and estrogen treatments.     

Distinct molecular regulation of glycogen synthase kinase-3alpha isozyme controlled by its N-terminal region: functional role in calcium/calpain signaling

Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and β. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3β, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of β-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/β-catenin pathway.     

Regulation of Tight Junction Resistance in T84 Monolayers by Elevation in Intracellular Ca2+: A Protein Kinase C Effect

Elevation in intracellular Ca²⁺ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T₈₄ cells, a human colon cancer line and a model Cl⁻ secretory epithelial cell. The Ca²⁺ ionophore A23187, which was used to increase the intracellular Ca²⁺ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na⁺/mannitol serosal-to-mucosal flux analysis performed across the T₈₄ monolayers treated with 2 μm A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between the Na⁺ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease in tight junction resistance. Whereas there was no effect of 0.1 μm A23187, 1 or 2 μm produced a 55% decrease in baseline resistance in 1 hr and 10 μm decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by washing 3 times with a Ringer's-HCO₃ solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca²⁺; loading the T₈₄ cells with the intracellular Ca²⁺ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca²⁺ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the action of A23187 on tight junction resistance, the Ca²⁺/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it had an additional effect in addition to mimicking the effect of elevating Ca²⁺. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 μm A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring. The results indicate that elevation in intracellular Ca²⁺ decreases tight junction resistance in the T₈₄ monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin ring. Received: 14 July 1995/Revised: 25 September 1995