BBA - Molecular Cell Research (v.1590, #1-3)

Transforming growth factor-β1 regulation of growth zone chondrocytes is mediated by multiple interacting pathways by Enrique Rosado; Zvi Schwartz; Victor L Sylvia; David D Dean; Barbara D Boyan (1-15).
Transforming growth factor beta 1 (TGF-β1) affects growth plate chondrocytes through Smad-mediated mechanisms and has been shown to increase protein kinase C (PKC). This study determined if PKC mediates the physiological response of rat costochondral growth zone (GC) chondrocytes to TGF-β1; if the physiological response occurs via type II or type III TGF-β receptors, and, if so, which receptor mediates the increase in PKC; and the signal transduction pathways involved. Treatment of confluent GC cells with TGF-β1 stimulated [3H]thymidine and [35S]sulfate incorporation as well as alkaline phosphatase (ALPase) and PKC specific activities. Inhibition of PKC with chelerythrine, staurosporine, or H-7 caused a dose-dependent decrease in these parameters, indicating that PKC signaling was involved. TGF-β1-dependent PKC and the physiological response of GC cells to TGF-β1 was reversed by anti-type II TGF-β receptor antibody and soluble type II TGF-β receptor, showing that TGF-β1 mediates these effects through the type II receptor. The increase in [3H]thymidine incorporation and ALPase specific activity were also regulated by protein kinase A (PKA) signaling, since the effects of TGF-β1 were partially blocked by the PKA inhibitor H-8. The mechanism of TGF-β1 activation of PKC is through phospholipase A2 (PLA2) and not through phospholipase C (PLC). Arachidonic acid increased PKC in control cultures and was additive with TGF-β1. Prostanoids are required, as indomethacin blocked the effect of TGF-β1, and Cox-1, but not Cox-2, is involved. TGF-β1 stimulates prostaglandin E2 (PGE2) production and exogenous PGE2 stimulates PKC, but not as much as TGF-β1, suggesting that PGE2 is not sufficient for all of the prostaglandin effect. In contrast, TGF-β1 was not regulated by diacylglycerol; neither dioctanoylglycerol (DOG) nor inhibition of diacylglycerol kinase with R59022 had an effect. G-proteins mediate TGF-β1 signaling at different levels in the cascade. TGF-β1-dependent increases in PGE2 levels and PKC were augmented by the G protein activator GTPγS, whereas inhibition of G-protein activity via GDPβS, pertussis toxin, or cholera toxin blocked stimulation of PKC by TGF-β1, indicating that both Gi and Gs are involved.Inhibition of PKA with H-8 partially blocked TGF-β1-dependent PKC, suggesting that PKA inhibition on the physiological response was via PKA regulation of PKC signaling. This indicates that multiple interacting signaling pathways are involved: TGF-β1 stimulates PLA2 and prostaglandin release via the action of Cox-1 on arachidonic acid. PGE2 activates the EP2 receptor, leading to G-protein-dependent activation of PKA. PKA signaling results in increased PKC activity and PKC signaling regulates proliferation, differentiation, and matrix synthesis.
Keywords: Chondrocyte culture; Transforming growth factor-β1; 1,25-(OH)2D3; Protein kinase C; Alkaline phosphatase; Signal transduction;

Participation of the conventional calpains in apoptosis by Tao Lu; Ying Xu; Maura T. Mericle; Ronald L. Mellgren (16-26).
The conventional calpains, m- and μ-calpain, are suggested to be involved in apoptosis triggered by many different mechanisms. However, it has not been possible to definitively associate calpain function with apoptosis, largely because of the incomplete selectivity of the cell permeable calpain inhibitors used in previous studies. In the present study, Chinese hamster ovary (CHO) cell lines overexpressing μ-calpain or the highly specific calpain inhibitor protein, calpastatin, have been utilized to explore apoptosis signals that are influenced by calpain content. This approach allows unambiguous alteration of calpain activity in cells. Serum depletion, treatment with the endoplasmic reticulum (ER) calcium ATPase inhibitor thapsigargin, and treatment with calcium ionophore A23187 produced apoptosis in CHO cells, which was increased in calpain overexpressing cells and decreased by induced expression of calpastatin. Inhibition of calpain activity protected β-spectrin, but not α-spectrin, from proteolysis. The calpains seemed not to be involved in apoptosis triggered by a number of other treatments. Calpain protected against TNF-α induced apoptosis. In contrast to previous studies, we found no evidence that calpains proteolyze IκB-α in TNF-α-stimulated cells. These studies indicate that the conventional calpains participate in some, but not all, apoptotic signaling mechanisms. In most cases, they contributed to apoptosis, but in at least one case, they were protective.
Keywords: Calpain; Apoptosis; Thapsigargin; A23187; TNF-alpha; CHO cell;

Characterisation of two 14-3-3 genes from Trichoderma reesei: interactions with yeast secretory pathway components by Tuija Vasara; Sirkka Keränen; Merja Penttilä; Markku Saloheimo (27-40).
The 14-3-3 proteins are highly conserved, ubiquitously expressed proteins taking part in numerous cellular processes. Two genes encoding 14-3-3 proteins, ftt1 and ftt2, were isolated and characterised from the filamentous fungus Trichoderma reesei. FTTI showed the highest sequence identity (98% at the amino acid level) to the Trichoderma harzianum protein Th1433. FTTII is relatively distinct from FTTI, showing approximately 75% identity to other fungal 14-3-3 proteins. Despite their sequence divergence, both of the T. reesei ftt genes were equally able to complement the yeast bmh1 bmh2 double disruption. The T. reesei ftt genes were also found to be quite closely linked in the genomic DNA. A C-terminally truncated version of ftt1 (ftt1ΔC) was first isolated as a multicopy suppressor of the growth defect of the temperature-sensitive yeast secretory mutant sec15-1. Overexpression of ftt1ΔC also suppressed the growth defect of sec2-41, sec3-101, and sec7-1 strains. Overexpression of ftt1ΔC in sec2-41 and sec15-1 strains could also rescue the secretion of invertase at the restrictive temperatures, and overexpression of full-length ftt1 enhanced invertase secretion by wild-type yeast cells. These findings strongly suggest that the T. reesei ftt1 has a role in protein secretion.
Keywords: 14-3-3; Trichoderma reesei/Hypocrea jecorina; Exocytosis;

Activation of a Src-dependent Raf–MEK1/2–ERK signaling pathway is required for IL-1α-induced upregulation of β-defensin 2 in human middle ear epithelial cells by Sung-Kyun Moon; Haa-Yung Lee; Jian-Dong Li; Mitsuyoshi Nagura; Sung-Ho Kang; Young-Myoung Chun; Fred H Linthicum; Tomas Ganz; Ali Andalibi; David J Lim (41-51).
β-defensin 2 is produced by a variety of epithelial cell types in the body and exhibits potent antimicrobial activity against a variety of pathogens, including the bacteria that are most commonly associated with otitis media (OM). The human β-defensin 2 (hBD-2) gene is an NF-κB regulated gene and a variety of proinflammatory stimuli can induce its expression. Although the presence of molecules of innate immunity such as lysozyme and lactoferrin has been demonstrated in the middle ear, to date there have been no reports on the expression of β-defensin 2. In the present study, we demonstrate that β-defensin 2 is expressed in the middle ear mucosa of humans and rats. We also show that it is expressed in a human middle ear epithelial cell line and that its expression is induced by proinflammatory stimuli such as interleukin 1 alpha (IL-1α), tumor necrosis factor alpha (TNF-α), and lipopolysaccharide (LPS). Moreover, we demonstrate that the transcriptional activation of hBD-2 gene by IL-1α is mediated through an Src-dependent Raf–MEK1/2–ERK signaling pathway.
Keywords: Src-dependent; β-defensin 2; Middle ear epithelial cell;

It has been reported that muscles of myoD−/− mice present a lower potential to regenerate, but there are no reports on the effect of acute interference with myoD expression limited in space and time to only a particular regenerating muscle. Here we relied on antisense inhibition of this factor. Four different oligos were tested. The suppression of regeneration indices (the expression of desmin, the formation of myotubes and the initiation of endplates) was the most pronounced, with the oligomer targeting a region encompassing the translation start site of myoD. A mixed backbone phosphorothioate–phosphate diester oligo (200 μl at 20 μM) was still detectable in the muscles 1 h after its administration and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the level of the targeted 5′ end of the myoD mRNA was selectively decreased. The level of myoD protein was also lowered. Four hours after the antisense treatment, when the oligos were no longer detectable, the myoD mRNA level was restored and 24 h later it exceeded controls together with that of myf-5 and myogenin. After 4 weeks, the antisense-treated soleus muscles were similar to the control-treated and the untreated regenerated soleus with respect to fiber types and motor endplates, however, they contained smaller fibers which reflected the asynchronity of regeneration. This shows that successfully targeted simple antisense oligonucleotides can be used as selective tools for inhibition of individual factors in studying the process of muscle regeneration.
Keywords: Antisense oligonucleotide; myoD; Muscle regeneration;

Role of human organic anion transporter 4 in the transport of ochratoxin A by Ellappan Babu; Michio Takeda; Shinichi Narikawa; Yukari Kobayashi; Atsushi Enomoto; Akihiro Tojo; Seok Ho Cha; Takashi Sekine; Dhanapal Sakthisekaran; Hitoshi Endou (64-75).
The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporter 4 (hOAT4) using mouse proximal tubule cells stably transfected with hOAT4 (S2 hOAT4). Immunohistochemical analysis revealed that hOAT4 protein was localized to the apical side of the proximal tubule. S2 hOAT4 expressed hOAT4 protein in the apical side as well as basolateral side and the cells were cultured on the plastic dish for experiments. S2 hOAT4 exhibited a time- and concentration-dependent, and a saturable increase in OTA uptake, with an apparent K m value of 22.9±2.44 μM. The OTA uptakes were inhibited by several substrates for the OATs. Probenecid, piroxicam, octanoate and citrinin inhibited OTA uptake by hOAT4 in a competitive manner (K i=44.4–336.4 μM), with the following order of potency: probenecid>piroxicam>octanoate>citrinin. The efflux of OTA by S2 hOAT4 was higher than that by mock. Addition of OTA resulted in slight decrease in viability of S2 hOAT4 compared with mock. These results indicate that hOAT4 mediates the high-affinity transport of OTA on the apical side of the proximal tubule, whereas the transport characteristics of OTA are distinct from those by basolateral OATs.
Keywords: Ochratoxin A; Organic anion transporter; Proximal tubule; Transport; Cell line;

Nanomolar concentrations of Taxol, and other antimitotic agents that interact with microtubules, mediate serine phosphorylation of the 66-kDa Shc isoform (p66shc) in A549 human lung carcinoma cells, 9–18 h after drug treatment. This event coincides with the release of PARP cleavage fragments that are early indicators of apoptosis. Taxol-induced serine phosphorylation of p66shc results from a MEK-independent signaling pathway that is activated in A549 cells that have a prolonged or abnormal mitotic phase of the cell cycle [Cancer Res. 60 (2000) 5171]. In contrast, in murine macrophage RAW 264.7 cells, micromolar concentrations of Taxol but not other microtubule-interacting agents induced serine phosphorylation of p66shc that correlated with the phosphorylation of Raf-1 and extracellular signal-regulated kinase (ERK1/2), within 15–30 min after Taxol treatment. This event also was induced by lipopolysaccharide (LPS). The MEK-inhibitor, U0126, that specifically inhibits the activation of ERK also blocked the phosphorylation of p66shc and Raf-1, suggesting that these processes were MEK-dependent, quite different from that which was observed in A549 cells. Taxol also induced phosphorylation of p38 and JNK MAP kinases within 8–15 min after drug treatment. It is known that Taxol, but not other microtubule-interacting agents, induces the production of cytokines, such as tumor necrosis factor alpha (TNF-α) in mouse macrophages. The time course of Taxol-induced TNF-α expression coincides with that of Taxol-induced p66shc phosphorylation, and U0126 inhibits significantly Taxol-induced TNF-α expression in RAW 264.7 cells. Our data indicate that the Taxol-induced serine phosphorylation of p66shc in RAW 264.7 cells is microtubule-independent and may be related to increased TNF-α expression after Taxol and LPS treatment. It is concluded that the mechanisms involved in Taxol-induced p66shc phosphorylation are distinct in A549 and RAW 264.7 cells.
Keywords: Taxol; p66shc phosphorylation; Microtubule-interacting agent; Macrophage;

This study investigates the regulation of cAMP-stimulated casein secretion in rat mammary explants by cAMP phosphodiesterase (cAMP-PDE) activity. cAMP-PDE activity of the lactating rat mammary gland is shown to be provided by three families, types II, III and IV. In mammary explants, general inhibition of the cAMP-PDE activity significantly increased the rate of cAMP-stimulated casein secretion. This effect could be mimicked using the type-IV specific inhibitor rolipram but not by the specific, or combined, inhibition of the type II and type III activity. Only type IV activity significantly affected intracellular accumulation of cAMP whereas all three cAMP-PDE activities were shown to influence the PKA activation ratio in cells. RtPCR analysis showed that the mammary gland apparently expresses just three type IV isozymes, RNPDE4A5, RNPDE4A8 and RNPDE4D3. A specific role for type IV cAMP-PDE activity in the regulation of casein secretion is suggested and possible mechanisms for the effects of PDEIV activity discussed.
Keywords: Mammary; Casein; Secretion; Phosphodiesterase; cAMP; PDEIV;

Interaction of S-adenosylhomocysteine hydrolase of Xenopus laevis with mRNA(guanine-7-)methyltransferase: implication on its nuclear compartmentalisation and on cap methylation of hnRNA by Norbert Radomski; Guillermo Barreto; Christine Kaufmann; Jun'ichi Yokoska; Kiyohisa Mizumoto; Christine Dreyer (93-102).
S-adenosylhomocysteine hydrolase (SAHH) is the only enzyme known to cleave S-adenosylhomocysteine (SAH), a product and an inhibitor of all S-adenosylmethionine-dependent transmethylation reactions. Xenopus SAHH is a nuclear enzyme in transcriptionally active cells and inhibition of xSAHH prevents cap methylation of hnRNA [Mol. Biol. Cell 10 (1999) 4283]. Here, we demonstrate that inhibition of xSAHH in Xenopus XTC cells results in a cytoplasmic accumulation of the shuttling hnRNPs, while xSAHH itself remains in the nucleus. The functional link between xSAHH and mRNA cap methylation is further supported by a physical association between xSAHH and mRNA(guanine-7-)methyltransferase (CMT). We show by co-immunoprecipitation of tagged proteins that both enzymes interact in vivo. Direct interaction in vitro is shown by pull-down experiments that further demonstrate that the N-terminal 55 amino acids of xSAHH are sufficient for binding to CMT. Since CMT is known to bind to the hyperphoshorylated C-terminal domain (CTD) of its large subunit of RNA polymerase II, we have studied the co-localisation of RNA polymerase II and xSAHH in oocyte nuclei. Immunolocalisation on spreads of lampbrush chromosomes shows xSAHH on the loops of the transcriptionally active lampbrush chromosomes, in Cajal bodies and in B-snurposomes, the nuclear compartments that are most likely engaged in storage and recycling of RNA polymerase II and its cofactors. We therefore suggest that a subfraction of the nuclear xSAHH remains associated with the RNA polymerase holoenzyme complexes, also while these are not actively engaged in transcription.
Keywords: S-adenosylhomocysteine hydrolase; mRNA(guanine-7-)methyltransferase (CMT); hnRNA; Cajal body; B-snurposome; Xenopus oocyte;

CHO-K1 cells were synchronized at the G1/S border by mitotic shake-off and aphidicolin incubation. Pulse-labeling with tritium was done at 30 min, 2 or 5 h into the S-phase, with chase incubations for different times in non-radioactive medium. The cells were subjected to neutral microelectrophoresis to extend the DNA into “comets,” after which the label was visualized through autoradiography. At zero chase time, all label was positioned in the head. The displacement of label into the tails increased with time, reaching a maximum at about 5 h after the pulse. A lag phase of 2–3 h was observed for the early-labeled cells before the displacement started. Also, more label was released after overnight serum starvation, but this was reversed through a 3-h incubation at normal growth conditions. It was found that late-replicating chromatin is organized in larger domains than early-replicating chromatin, and DNA polymerase seems to be an important organizer. Early-replicating chromatin has other important attachments to the nuclear matrix, dependent on metabolic activity.
Keywords: Chromatin; Comet; Single-cell electrophoresis; Domain; Loop; Replication;

A calcium-permeable channel activated by muscarinic acetylcholine receptors and InsP3 in developing chick ciliary ganglion neurons by Carla Distasi; Federico Di Gregorio; Alessandra Gilardino; Davide Lovisolo (109-122).
The electrical responses elicited by the muscarinic cholinergic pathway have been studied in cultured embryonic chick ciliary ganglion (CG) neurons. Neurons obtained from E7–E8 ganglia were maintained in serum-free medium for 1 to 3 days. Stimulation with 50 μM muscarine induced depolarizing responses in about 30% of the cells tested. In voltage clamp experiments at a holding potential of −50 mV, an inward current could be recorded in the same percentage of cells in response to muscarinic stimulation. In single channel experiments, with standard physiological solution in the pipette, muscarine transiently activated an inward conducting channel. Cell-attached recordings with 100 mM CaCl2 in the pipette provided evidence that muscarinic agonists can activate a cationic calcium-permeable channel. Two main conductance levels could be detected, of 2.3±0.6 and 5.6±0.6 pS, respectively. In excised patches, addition of 5–20 μM inositol 1,4,5-trisphosphate (InsP3) to the bath reactivated a channel that could be blocked by heparin and whose characteristics were very similar to those of the channel seen in response to muscarinic stimulation. A channel with similar properties has been previously shown to be activated by basic fibroblast growth factor (bFGF) and InsP3 in the same preparation.
Keywords: Muscarinic acetylcholine receptor; Calcium-permeable channel; InsP3; Chick embryo neuron; Ciliary ganglion;

Inhibition of Rho/Rho-kinase signaling downregulates plasminogen activator inhibitor-1 synthesis in cultured human monocytes by Toshiyuki Ishibashi; Kenji Nagata; Hiroshi Ohkawara; Takayuki Sakamoto; Keiko Yokoyama; Joji Shindo; Koichi Sugimoto; Sotaro Sakurada; Yoh Takuwa; Tamio Teramoto; Yukio Maruyama (123-130).
Increased production of plasminogen activator inhibitor-1 (PAI-1) in plaques plays a role in the pathogenesis of atherosclerosis. This study was conducted to investigate the effect of blockade of Rho/Rho-kinase signaling on the synthesis of PAI-1 in cultured human peripheral blood monocytes. HMG-CoA reductase inhibitors (statins) and inhibitors of Rho and Rho-kinase were added to monocyte cultures. The levels of PAI antigen and mRNA were determined by Western blotting and RT-PCR, respectively, and PAI-1 expression was assessed by immunohistochemistry. We performed pull-down assays to determine the activity of Rho by measuring the GTP-bound form of Rho A. In unstimulated and lipopolysaccharide (LPS)-stimulated cultured monocytes, statins reduced the levels of PAI-1 antigen and mRNA. The suppressive effects of statins on PAI-1 synthesis were reversed by geranylgeranylpyrophosphate (GGPP) and were mimicked by C3 exoenzyme. Immunohistochemistry confirmed the role of lipid modification by GGPP in suppressive effect of statins in PAI-1 synthesis. Pull-down assays demonstrated that statins decreased the levels of the GTP-bound form of Rho A. Our findings suggest that statins decrease the activity of Rho by inhibiting geranylgeranylation. Moreover, Rho-kinase inhibitors, Y-27632 and fasudil, suppressed the synthesis of PAI-1 in this culture system. We show that inhibition of Rho/Rho-kinase signaling downregulates the synthesis of PAI-1 in human monocytes.
Keywords: Plasminogen activator inhibitor-1; Statin; Rho; Geranylgeranylation; Rho-kinase;

A two-step approach is described to chemically camouflage the inert surface of model polystyrene nanospheres of 60 nm in diameter against recognition by the body's defenses. The first step was based on the strong protein adsorbing potency of polystyrene, and the second step utilized the chemical reactivity of the adsorbed proteins for conjugation with cyanuric chloride-activated methoxypoly(ethyleneglycol)5000, mPEG5000. Bovine serum albumin (BSA) and rat IgG were used as models of non-immune and immune proteins, respectively. The maximum adsorbance values for both proteins were near expectation for a close-packed monolayer. Adsorption isotherms studies and analysis of the hydrodynamic thickness of the adsorbed protein layer confirmed the close-packed side-on mode of adsorption for BSA and the end-on mode of adsorption for IgG, respectively. Nucleophiles on the adsorbed proteins were then reacted with cyanuric chloride activated mPEG5000. The average poly(ethyleneglycol) (PEG) content for a 60-nm nanospheres was found to be 13.7±0.4 μmol PEG/μmol BSA and 3.6±0.3 μmol PEG/μmol IgG. The interaction of both PEG-bearing nanospheres with the hydrophobic column material octyl-agarose indicated surface heterogeneity among the nanospheres. Only nanospheres with the most hydrophilic phenotype (approximately 70% of the total population) exhibited stealth properties after intravenous injection to rats. In contrast to the described approach, incubation of uncoated nanospheres with preformed BSA-mPEG5000 conjugates failed to produce long circulating entities. For design of splenotropic particles cyanuric chloride-activated mPEG5000 was conjugated to BSA-coated polystyrene beads of 225 nm in diameter. Despite their stealth property to hepatic Kupffer cell recognition, these nanospheres were cleared by the splenic red pulp macrophages.
Keywords: Poly(ethyleneglycol); Nanosphere; Macrophage; Kupffer cell; Protein adsorption; Hydrophobic interaction chromatography;

Recently, the presence of 2′,5′-linked oligoadenylates and a high 2′,5′-oligoadenylate synthetase activity were discovered in a lower invertebrate, the marine sponge Geodia cydonium. It has been demonstrated that mammalian 2–5A synthetase isozymes require a dsRNA cofactor for their enzymatic activity. Our results show that, unlike mammalian 2–5A synthetases, the 2–5A synthetase from the sponge acts in a dsRNA-independent manner in vitro. A prolonged incubation of the G. cydonium extract with a high concentration of a micrococcal nuclease had no effect on the activity of the 2–5A synthetase. At the same time, the micrococcal nuclease was effective within 30 min in degrading dsRNA needed for the enzymatic activity in IFN-induced PC12 cells. These results indicate that the 2–5A synthetase from G. cydonium may be active per se or is activated by some other mechanism. The sponge enzyme is capable of synthesizing a series of 2–5A oligomers ranging from dimers to octamers. The accumulation of a dimer in the predominant proportion during the first stage of the reaction was observed, followed by a gradual increase in longer oligoadenylates. By its product profile and kinetics of formation, the sponge 2–5A synthetase behaves like a specific isoform of enzymes of the 2–5A synthetase family.
Keywords: 2–5A synthetase; Sponge; Geodia cydonium; Enzymatic activity; dsRNA;

Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes by Sai Wah Tsao; Xianghong Wang; Yu Liu; Yuk Chun Cheung; Huichen Feng; Zhong Zheng; Natalie Wong; P.W Yuen; Angela K.F Lo; Y.C Wong; D.P Huang (150-158).
Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially in southern China. One of the most striking features of this disease is its close relationship with Epstein–Barr Virus (EBV). However, to date there is no direct study on the mechanisms involved in the role of EBV in the tumorigenesis of NPC, largely due to lack of an experimental model. Available hypotheses on the association between EBV and NPC are generated from non-nasopharyngeal epithelial cell systems such as human keratinocytes or mouse epithelial cells, which may not truly represent the biological properties of nasopharyngeal epithelial (NP) cells. In this study, we report the establishment of two immortalized NP cell lines, NP69SV40T and NP39E6/E7, using SV40T and HPV16E6/E7 oncogenes. We found that NP60SV40T and NP39E6/E7 cell lines not only maintained many characteristics of normal NP cells (i.e. keratin profile and responsive to TGFβ inhibition) but also highly responsive to one of the EBV encoded genes, LMP1. Comparative genome hybridization (CGH) analysis showed that these two cell lines contained multiple genetic alterations, some of which have been described in NPC. The immortalized NP cell lines are non-tumorigenic and exhibit anchorage-dependent growth. These cell lines may provide a possible cell model system for studying the mechanisms involved in the tumorigenesis of NPC.
Keywords: Nasopharyngeal epithelial; HPVE6/E7; SV40T;

Synaptotagmin II could confer Ca2+ sensitivity to phagocytosis in human neutrophils by I.Maria Lindmark; Anna Karlsson; Lena Serrander; Patrice Francois; Daniel Lew; Birgitta Rasmusson; Olle Stendahl; Oliver Nüße (159-166).
Phagolysosome fusion and granule exocytosis in neutrophils are calcium-dependent processes. The calcium requirements vary between granule types, suggesting the presence of different calcium sensors. The synaptotagmins, a family of calcium-binding proteins, previously shown to participate in vesicle fusion and vesicle recycling in excitable cells, are putative calcium-sensors of exocytosis in excitable cells. In this study, we show that synaptotagmin II is present in human neutrophils and may participate in phagocytic and in exocytotic processes. In protein extracts from human neutrophils, we identified synaptotagmin II by Western blot as an 80 kDa protein. Subcellular fractionation revealed that synaptotagmin II was associated with the specific granules. In fMLP-stimulated cells, synaptotagmin II translocated to the plasma membrane. This correlated with the upregulation of complement receptor 3 (CR 3), reflecting the translocation of specific granules to the cell surface. Synaptotagmin II also translocated to the phagosome after complement-mediated phagocytosis in the presence of calcium. LAMP-1 translocated in parallel but probably was located to another subcellular compartment than synaptotagmin II. Under calcium-reduced conditions, neither synaptotagmin II nor LAMP-1 translocated to the phagosome. We therefore suggest a role for synaptotagmin II as calcium-sensor during phagocytosis and secretion in neutrophils.
Keywords: PMN; Phagocytosis; Secretion; Synaptotagmin; Secondary granule;

Regulation of TIMP-1 phenotypic expression in Epstein–Barr virus-immortalized B lymphocytes by Candice Trocmé; Philippe Gaudin; Sylvie Berthier; Françoise Morel (167-176).
Normal B lymphocytes as well as malignant B cells extravasate from blood circulation during physiological and pathological processes and require matrix metalloproteinases (MMPs) to facilitate trafficking through the subendothelial basal lamina and the extracellular matrix. We have previously shown that Epstein–Barr virus (EBV)-immortalized B lymphocytes constitutively synthesized low levels of MMP-9 and huge amounts of its preferential inhibitor, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). In the present study, TIMP-1 phenotypic expression was extensively investigated in response to various mediators including interleukins, chemokines, growth factors and tumor promotor, and was compared to MMP-9 synthesis. Results showed a roughly constitutive TIMP-1 expression opposed to an inducible MMP-9 synthesis. Nevertheless, further analysis of TIMP-1 synthesis showed the existence of regulation mechanisms: modulation of intracellular Ca2+ concentration as well as cation ionophore monensin were demonstrated to influence TIMP-1 production and secretion. The precise pathways implicated in these regulation mechanisms are currently under survey.
Keywords: B lymphocyte; Epstein–Barr virus; Phenotypic regulation; TIMP; Matrix metalloproteinase; Ion movement;

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex—comprising precursor, hsp70 and 14–3–3 proteins, as well as several unidentified components—docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.
Keywords: Chloroplast protein import; Translocon; Toc apparatus; Tic apparatus; Chloroplast envelope; Protein targeting;