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I Schulz

Publications and source records attributed to I Schulz.

At least 19 recordsLinked to original sources

The Arabidopsis thaliana genome contains at least 29 active genes encoding SET domain proteins that can be assigned to four evolutionarily conserved classes.

SET domains are conserved amino acid motifs present in chromosomal proteins that function in epigenetic control of gene expression. These proteins can be divided into four classes as typified by their Drosophila members E(Z), TRX, ASH1 and SU(VAR)3-9. Homologs of all four classes have been identified in yeast and mammals, but not in plants. A BLASTP screening of the Arabidopsis genome identified 37 genes: three E(z) homologs, five trx homologs, four ash1 homologs and 15 genes similar to Su(var)3-9. Seven genes were assigned as trx-related and three as ash1-related. Only four genes have been described previously. Our classification is based on the characteristics of the SET domains, cysteine-rich regions and additional conserved domains, including a novel YGD domain. RT-PCR analysis, cDNA cloning and matching ESTs show that at least 29 of the genes are active in diverse tissues. The high number of SET domain genes, possibly involved in epigenetic control of gene activity during plant development, can partly be explained by extensive genome duplication in Arabidopsis. Additionally, the lack of introns in the coding region of eight SU(VAR)3-9 class genes indicates evolution of new genes by retrotransposition. The identification of putative nuclear localization signals and AT-hooks in many of the proteins supports an anticipated nuclear localization, which was demonstrated for selected proteins.

Active Transport, Cell Nucleus↗

Ventilator-associated pneumonia in a surgical intensive care unit: epidemiology, etiology and comparison of three bronchoscopic methods for microbiological specimen sampling.

BACKGROUND: Ventilator-associated bacterial pneumonia (VAP) is a important intensive care unit (ICU)-acquired infection in mechanically ventilated patients. Early and correct diagnosis of VAP is difficult but is an urgent challenge for an optimal antibiotic treatment. The aim of the study was to evaluate the incidence and microbiology of ventilator-associated pneumonia and to compare three quantitative bronchoscopic methods for diagnosis. METHODS: A prospective, open, epidemiological clinical study was performed in a surgical ICU. In a prospective study, 279 patients admitted to a 14-bed surgical ICU during a 1-year period were evaluated with regard to VAP. Three quantitative culture bronchoscopic techniques for identifying the etiological agent were compared [bronchoalveolar lavage (BAL), protected specimen brush (PSB) and bronchoscopic tracheobronchial secretion (TBS)]. RESULTS: Among 103 long-term ventilated patients, 49 (48%) developed one or more VAPs (a total of 60 VAPs). The incidence was 24 VAPs per 100 ventilated patients or 23 VAPs per 1000 ventilator days. BAL, PSB and TBS with quantitative measurements were equivalent in identifying the bacterial etiology. The VAP was caused predominantly by Staphylococcus aureus in 38% of cases, followed by Pseudomonas aeruginosa in 10%, Haemophilus influenzae in 10% and Klebsiella sp. in 9%. We did not find an increased mortality rate in patients undergoing long-term ventilation who acquired VAP in comparison with patients without VAP. CONCLUSION: For the identification of the microbiological etiology of VAP, one of three available bronchoscopic methods analysed by quantitative measurements is sufficient. In our study, quantitative bronchoscopic tracheal secretion analysis was very promising. Before accepting this method as a standard technique, other studies will have to confirm our results.

Bronchoscopy↗

Arachidonic acid modulates the spatiotemporal characteristics of agonist-evoked Ca2+ waves in mouse pancreatic acinar cells.

In pancreatic acinar cells analysis of the propagation speed of secretagogue-evoked Ca2+ waves can be used to examine coupling of hormone receptors to intracellular signal cascades that cause activation of protein kinase C or production of arachidonic acid (AA). In the present study we have investigated the role of cytosolic phospholipase A2 (cPLA2) and AA in acetylcholine (ACh)- and bombesin-induced Ca2+ signaling. Inhibition of cPLA2 caused acceleration of ACh-induced Ca2+ waves, whereas bombesin-evoked Ca2+ waves were unaffected. When enzymatic metabolization of AA was prevented with the cyclooxygenase inhibitor indomethacin or the lipoxygenase inhibitor nordihydroguaiaretic acid, ACh-induced Ca2+ waves were slowed down. Agonist-induced activation of cPLA2 involves mitogen-activated protein kinase (MAPK) activation. An increase in phosphorylation of p38(MAPK) and p42/44(MAPK) within 10 s after stimulation could be demonstrated for ACh but was absent for bombesin. Rapid phosphorylation of p38(MAPK) and p42/44(MAPK) could also be observed in the presence of cholecystokinin (CCK), which also causes activation of cPLA2. ACh-and CCK-induced Ca2+ waves were slowed down when p38(MAPK) was inhibited with SB 203580, whereas inhibition of p42/44(MAPK) with PD 98059 caused acceleration of ACh- and CCK-induced Ca2+ waves. The spreading of bombesin-evoked Ca2+ waves was affected neither by PD 98059 nor by SB 203580. Our data indicate that in mouse pancreatic acinar cells both ACh and CCK receptors couple to the cPLA2 pathway. cPLA2 activation occurs within 1-2 s after hormone application and is promoted by p42/44(MAPK) and inhibited by p38(MAPK). Furthermore, the data demonstrate that secondary (Ca2+-induced) Ca2+ release, which supports Ca2+ wave spreading, is inhibited by AA itself and not by a metabolite of AA.

Acetylcholine↗

tert-Butoxyl radicals generate mainly 7,8-dihydro-8-oxoguanine in DNA.

Like hydroxyl radicals, alkoxyl radicals have been implicated in the generation of cellular oxidative DNA damage under physiological conditions; however, their genotoxic potential has not yet been established. We have analyzed the DNA damage induced by a photochemical source of tert-butoxyl radicals, the water soluble peroxy ester [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), using various repair endonucleases as probes. The irradiation (UV(360)) of BCBT in the presence of bacteriophage PM2 DNA was found to generate a DNA damage profile that consisted mostly of base modifications sensitive to the repair endonuclease Fpg protein. Approximately 90% of the modifications were identified as 7,8-dihydro-8-oxoguanine (8-oxoGua) residues by HPLC/ECD analysis. Oxidative pyrimidine modifications (sensitive to endonuclease III), sites of base loss (AP sites) and single-strand breaks were only minor modifications. Experiments with various scavengers and quenchers indicated that the DNA damage by BCBT+UV(360) was caused by tert-butoxyl radicals as the ultimate reactive species. The mutagenicity associated with the induced damage was analyzed in the gpt gene of plasmid pSV2gpt, which was exposed to BCBT+UV(360) and subsequently transfected into Escherichia coli. The results were in agreement with the specific generation of 8-oxoGua. Nearly all point mutations (20 out of 21) were found to be GC-->TA transversions known to be characteristic for 8-oxoGua. In conclusion, alkoxyl radicals generated from BCBT+UV(360) induce 8-oxoGua in DNA with a higher selectivity than any other reactive oxygen species analyzed so far.

Alcohols↗

Multicentre evaluation of a new point-of-care test for the quantitative determination of D-dimer.

Imprecision studies, interference testing and multicentre method comparisons using patient samples were carried out with of a new point-of-care test for D-dimer (CARDIAC D-Dimer). The CV of the within-series and the day-to-day imprecision with blood samples and control materials were between 7% and 13%. Compared with Tina-quant D-Dimer, CARDIAC D-Dimer showed a good correlation and accuracy (n=353; r=0.91; y=1.06x-0.03), compared with STA LIATEST D-Dimer some poorer accuracy (n=304; r=0.91; y=1.12x-0.03). No interference was detected for different hematocrit values (16% to 51%) and in investigations with hemoglobin (up to 0.13 mmol/l), biotin (up to 30 microg/l), bilirubin (up to 340 micromol/l), intralipid (up to 31.1 mmol/l) and rheumatic factor (up to 79 IU/ml). Overdosing or underdosing by 10 microl did not affect the test result. The diagnostic sensitivity of CARDIAC D-Dimer for the detection of acute venous thromboembolic diseases was 100% in our study. With CARDIAC D-Dimer reliable quantitative D-dimer results can be easily obtained. Because of the good analytical and clinical agreement with Tina-quant D-Dimer, it should be suitable for ruling out venous thromboembolic diseases.

Clinical Chemistry Tests↗

IP(3)and cyclic ADP-ribose induced Ca(2+) release from intracellular stores of pancreatic acinar cells from rat in primary culture.

We have measured Ca(2+)concentration changes in intracellular Ca(2+)stores ([Ca(2+)](store)) of rat pancreatic acinar cells in primary culture in response to the Ca(2+)mobilizing substances inositol-1,4,5-trisphosphate (IP(3)) and cyclic ADP-ribose (cADPr) using the Ca(2+)-sensitive dye mag Fura-2. We found that in this cell model IP(3)releases Ca(2+)in a quantal manner. Higher Ca(2+)concentration in the stores allowed a response to lower IP(3)concentrations ([IP(3)]) indicating that the sensitivity of IP(3)receptors to IP(3)is regulated by the Ca(2+)concentration in the stores. Cyclic ADPr, that modifies 'Ca(2+)-induced-Ca(2+)-release' (CICR), was also able to release Ca(2+)from intracellular stores of pancreatic acinar cells in primary culture. In comparison to the Ca(2+)ionophore ionomycin, which induced a maximal decrease (100%) in [Ca(2+)](store), a hypermaximal [IP(3)] (10 microM) dropped [Ca(2+)](store)by 87% and cADPr had no further effect. Cyclic ADPr reduced [Ca(2+)](store)by only 56% and subsequent IP(3)addition caused further maximal decrease in [Ca(2+)](store). Furthermore, a maximal [IP(3)] caused the same decrease in [Ca(2+)](store)in all regions of the cell, whereas cADPr dropped the [Ca(2+)](store)between 20 and 80% in different cell regions. From these data we conclude that in primary cultured rat pancreatic acinar cells at least three types of Ca(2+)stores exist. One type possessing both cADPr receptors and IP(3)receptors, a second type possessing only IP(3)receptors, and a third type whose Ca(2+)can be released by ionomycin but neither by IP(3)nor by cADPr.

Adenosine Diphosphate Ribose↗

Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity.

Many fungal pathogens undergo a yeast-hyphal transition during their pathogenic development that requires rearrangement of the cytoskeleton, followed by directed membrane traffic towards the growth region. The role of microtubules and their dynamic behavior during this process is not well understood. Here we set out to elucidate the organization, cellular role and in vivo dynamics of microtubules in the dimorphic phytopathogen Ustilago maydis. Hyphae and unbudded yeast-like cells of U. maydis contain bundles of spindle pole body-independent microtubules. At the onset of bud formation two spherical tubulin structures focus microtubules towards the growth region, suggesting that they support polar growth in G(2), while spindle pole body-nucleated astral microtubules participate in nuclear migration in M and early G(1). Conditional mutants of an essential alpha-tubulin gene from U. maydis, tub1, confirmed a role for interphase microtubules in determination of cell polarity and growth. Observation of GFP-Tub1 fusion protein revealed that spindle pole body-independent and astral microtubules are dynamic, with elongation and shrinkage rates comparable to those found in vertebrate systems. In addition, very fast depolymerization was measured within microtubule bundles. Unexpectedly, interphase microtubules underwent bending and rapid translocations within the cell, suggesting that unknown motor activities participate in microtubule organization in U. maydis. Movies available on-line: http://www.biologists.com/JCS/movies/jcs1792.html

Base Sequence↗

Agonist-evoked mitochondrial Ca2+ signals in mouse pancreatic acinar cells.

In the present study we have investigated cytosolic and mitochondrial Ca(2+) signals in isolated mouse pancreatic acinar cells double-loaded with the fluorescent probes fluo-3 and rhod-2. Stimulation of pancreatic acinar cells with 500 nm acetylcholine caused release of Ca(2+) from intracellular stores and produced cytosolic Ca(2+) signals in form of Ca(2+) waves propagating from the luminal to the basal cell pole. The increase in the cytosolic Ca(2+) concentration was followed by Ca(2+) uptake into mitochondria. Between onset of cytosolic and mitochondrial Ca(2+) signals there was a delay of 10.7 +/- 0.4 s. Ca(2+) uptake into mitochondria could be inhibited with Ruthenium Red and carbonyl cyanide m-chlorophenylhydrazone, whereas 2,5-di-tert-butylhydroquinone, which inhibits sarco(endo)plasmic reticulum Ca(2+) ATPases, did not prevent Ca(2+) accumulation in mitochondria. Carbonyl cyanide m-chlorophenylhydrazone-induced Ca(2+) release from mitochondria could only be observed after a preceding stimulation of the cell with a physiological agonist or by treatment with 2, 5-di-tert-butylhydroquinone, indicating that under resting conditions mitochondria do not contain releasable Ca(2+) ions. Analysis of the propagation rate of acetylcholine-induced Ca(2+) waves revealed that inhibition of mitochondrial Ca(2+) uptake did not accelerate spreading of cytosolic Ca(2+) signals. Our experiments indicate that in the early phase of secretagogue-induced Ca(2+) signals, mitochondria behave as passive Ca(2+)-buffering elements and do not actively suppress spreading of Ca(2+) signals in pancreatic acinar cells.

Acetylcholine↗

Oxidative DNA base damage induced by singlet oxygen and photosensitization: recognition by repair endonucleases and mutagenicity.

We have analyzed the recognition by various repair endonucleases of DNA base modifications induced by three oxidants, viz. [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), a photochemical source of tert-butoxyl radicals, disodium salt of 1,4-etheno-2,3-benzodioxin-1,4-dipropanoic acid (NDPO(2)), a chemical source of singlet oxygen, and riboflavin, a type-I photosensitizer. The base modifications induced by BCBT, which were previously shown to be mostly 7,8-dihydro-8-oxoguanine (8-oxoGua) residues, were recognized by Fpg and Ogg1 proteins, but not by endonuclease IIII, Ntg1 and Ntg2 proteins. In the case of singlet oxygen induced damage, 8-oxoGua accounted for only 35% of the base modifications recognized by Fpg protein. The remaining Fpg-sensitive modifications were not recognized by Ogg1 protein and relatively poor by endonuclease III, but they were relatively good substrates of Ntg1 and Ntg2. In the case of the damage induced by photoexcited riboflavin, the fraction of Fpg-sensitive base modifications identified as 8-oxoGua was only 23%. In contrast to the damage induced by singlet oxygen, the remaining lesions were not only recognized by Ntg1 and Ntg2 proteins and (relatively poor) by endonuclease III, but also by Ogg1 protein. The analysis of the mutations observed after transfection of modified plasmid pSV2gpt into Escherichia coli revealed that all agents induced near exclusively GC-->TA and GC-->CG transversions, the numbers of which were correlated with the numbers of 8-oxoGua residues and Ntg-sensitive modifications, respectively. In conclusion, both singlet oxygen and the type-I photosensitizer riboflavin induce predominantly oxidative guanine modifications other than 8-oxoGua, which most probably give rise to GC-->CG transversions and in which eukaryotic cells are substrates of Ntg1 and Ntg2 proteins.

Bacterial Proteins↗

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation.

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis¿2-aminophenoxyĕthane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.

1-Butanol↗

Hormone-stimulated calcium release is inhibited by cytoskeleton-disrupting toxins in AR4-2J cells.

We have studied the role of the actin cytoskeleton in bombesin-induced inositol 1,4,5-trisphosphate (IP(3))-production and Ca(2+)release in the pancreatic acinar tumour cell line AR4-2J. Intracellular and extracellular free Ca(2+)concentrations were measured in cell suspensions, using Fura-2. Disruption of the actin cytoskeleton by pretreatment of the cells with latrunculin B (10 microM), cytochalasin D (10 microM) or toxin B from Clostridium difficile (20 ng/ml) for 5-29 h led to inhibition of both, bombesin-stimulated IP(3)-production and Ca(2+)release. The toxins had no effect on binding of bombesin to its receptor, on Ca(2+)uptake into intracellular stores and on resting Ca(2+)levels. Ca(2+)mobilization from intracellular stores, induced by thapsigargin (100 nM) or IP(3)(1 microM) was not impaired by latrunculin B. In latrunculin B-pretreated cells inhibition of both, bombesin-stimulated IP(3)- production and Ca(2+)release was partly suspended in the presence of aluminum fluoride, an activator of G-proteins. Aluminum fluoride had no effect on basal IP(3)and Ca(2+)levels of control and toxin-pretreated cells. We conclude that disruption of the actin cytoskeleton impairs coupling of the bombesin receptor to its G-protein, resulting in inhibition of phospholipase C-activity with subsequent decreases in IP(3)-production and Ca(2+)release.

Aluminum Compounds↗

Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network.

The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 microm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo. http://www. biologists.com/JCS/movies/jcs1334.html

Animals↗

Low cytoplasmic [Ca(2+)] activates I(CRAC) independently of global Ca(2+) store depletion in RBL-1 cells.

Release of Ca(2+) from inositol (1,4,5)-trisphosphate-sensitive Ca(2+) stores causes "capacitative calcium entry," which is mediated by the so-called "Ca(2+) release-activated Ca(2+) current" (I(CRAC)) in RBL-1 cells. Refilling of the Ca(2+) stores or high cytoplasmic [Ca(2+)] ([Ca(2+)](cyt)) inactivate I(CRAC). Here we address the question if also [Ca(2+)](cyt) lower than the resting [Ca(2+)](cyt) influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both I(CRAC) and [Ca(2+)] within Ca(2+) stores of RBL-1 cells ([Ca(2+)](store)). We found that low [Ca(2+)](cyt) in the range of 30-50 nM activates I(CRAC) and Ca(2+) influx spontaneously and independently of global Ca(2+) store depletion, while elevation of [Ca(2+)](cyt) to the resting [Ca(2+)](cyt) (100 nM) resulted in store dependence of I(CRAC) activation. We conclude that spontaneous activation of I(CRAC) by low [Ca(2+)](cyt) could serve as a feedback mechanism keeping the resting [Ca(2+)](cyt) constant.

Animals↗

Oxidative DNA damage and mutations induced by a polar photosensitizer, Ro19-8022.

The oxidative DNA damage induced by the polar photosensitizer Ro19-8022 in the presence of light was studied and correlated with the associated mutagenicity. Both in isolated DNA and AS52 Chinese hamster ovary cells, photoexcited Ro19-8022 gave rise to a DNA damage profile that was similar to that caused by singlet oxygen: base modifications sensitive to the repair endonuclease Fpg protein, which according to high-performance liquid chromatography (HPLC) analysis were predominantly 8-hydroxyguanine (8-oxoG) residues, were generated in much higher yield than single-strand breaks, sites of base loss (AP sites) and oxidative pyrimidine modifications sensitive to endonuclease III. Fifty percent of the Fpg-sensitive modifications were repaired within 2 h. Under conditions that induced 10 Fpg-sensitive modifications per 10(6) bp (six 8-oxoG residues per 10(6) bp), approximately 60 mutations per 10(6) cells were induced in the gpt locus of the AS52 cells. A rather similar mutation frequency was observed when a plasmid carrying the gpt gene was exposed to Ro19-8022 plus light under cell-free conditions and subsequently replicated in bacteria. Sequence analysis revealed that GC-->TA and GC-->CG transversions accounted for 90% of the base substitutions. A significant generation of micronuclei was detectable in AS52 cells exposed to the photosensitizer plus light as well.

Animals↗

Cholecystokinin-evoked Ca(2+) waves in isolated mouse pancreatic acinar cells are modulated by activation of cytosolic phospholipase A(2), phospholipase D, and protein kinase C.

We employed confocal laser-scanning microscopy to monitor cholecystokinin (CCK)-evoked Ca(2+) signals in fluo-3-loaded mouse pancreatic acinar cells. CCK-8-induced Ca(2+) signals start at the luminal cell pole and subsequently spread toward the basolateral membrane. Ca(2+) waves elicited by stimulation of high-affinity CCK receptors (h.a.CCK-R) with 20 pM CCK-8 spread with a slower rate than those induced by activation of low-affinity CCK receptors (l.a. CCK-R) with 10 nM CCK-8. However, the magnitude of the initial Ca(2+) release was the same at both CCK-8 concentrations, suggesting that the secondary Ca(2+) release from intracellular stores is modulated by activation of different intracellular pathways in response to low and high CCK-8 concentrations. Our experiments suggest that the propagation of Ca(2+) waves is modulated by protein kinase C (PKC) and arachidonic acid (AA). The data indicate that h.a. CCK-R are linked to phospholipase C (PLC) and phospholipase A(2) (PLA(2)) cascades, whereas l.a.CCK-R are coupled to PLC and phospholipase D (PLD) cascades. The products of PLA(2) and PLD activation, AA and diacylglycerol (DAG), cause inhibition of Ca(2+) wave propagation by yet unknown mechanisms.

Animals↗

Inhibition of amylase secretion from differentiated AR4-2J pancreatic acinar cells by an actin cytoskeleton controlled protein tyrosine phosphatase activity.

Disruption of the actin cytoskeleton in AR4-2J pancreatic acinar cells led to an increase in cytosolic protein tyrosine phosphatase activity, abolished bombesin-induced tyrosine phosphorylation and reduced bombesin-induced amylase secretion by about 45%. Furthermore, both tyrosine phosphorylation and amylase secretion induced by phorbol ester-induced activation of protein kinase C were abolished. An increase in the cytosolic free Ca2+ concentration by the Ca2+ ionophore A23187 had no effect on tyrosine phosphorylation but induced amylase release. Only when added together with phorbol ester, the same level of amylase secretion as with bombesin was reached. This amylase secretion was inhibited by about 40%, by actin cytoskeleton disruption similar to that induced by bombesin. We conclude that actin cytoskeleton-controlled protein tyrosine phosphatase activity downstream of protein kinase C activity regulates tyrosine phosphorylation which in part is involved in bombesin-stimulated amylase secretion.

Actins↗

Intracellular localization and in vivo trafficking of p24A and p23.

Recently, p24A and p23 (also termed Tmp21), two members of the p24 protein family, have been proposed to function as integral receptors for the COPI-vesicle coat. This study describes the intracellular localization and trafficking of p24A in comparison to p23. For immunolocalization of p24A and p23, strong reduction and denaturation conditions were necessary to allow antibody interaction. Both p24A and p23 cycle continuously between intermediate compartment (IC) elements and the cis-Golgi network. In vivo trafficking of p24A and p23 tagged to green fluorescent protein (GFP) revealed that both proteins travel by large (up to 1 micrometer in length) microtubule-dependent pre-Golgi carriers with a maximum speed of up to 1.6 micrometer s-1 from the IC to the Golgi cisternae. Aluminum fluoride, a general activator of heterotrimeric G-proteins, blocked peripheral pre-Golgi movements of GFP-p24A/p23 and inhibited fluorescence recovery after photobleaching in the perinuclear Golgi area. p24A and p23 are predominantly colocalized. Overexpression of GFP-p24A, to an extent which did not destroy the Golgi complex, induced delocalization of part of the proteins into ER elements. This study therefore gives new insights into the localization and trafficking behavior of the two COPI-binding proteins p24A and p23.

Aluminum Compounds↗

A comparative study of rat and human Tmp21 (p23) reveals the pseudogene-like features of human Tmp21-II.

Tmp21 (p23) is involved in biosynthetic transport from the endoplasmic reticulum to the Golgi complex. We have recently characterized two cDNA-variants of human Tmp21, the Tmp21-isoforms-I and -II. Because of the lack of cDNA sequence data and protein expression data, it was not clear if Tmp21-II encodes a functional Tmp21-protein. Here we describe the cloning of the full length human Tmp21-II transcript. The putative open reading frame of Tmp21-II contains a reading frame jump and a nonsense mutation in comparison to all other Tmp21-I (p23) members. Our data indicate that hum-Tmp21-II is transcribed, but not translated. We conclude that Tmp21-II cDNA derives from a neutral pseudogene, which originates from a duplication event of the human Tmp21-isoform-I.

Amino Acid Sequence↗