bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluating the risk of tuberous sclerosis in cases with prenatal diagnosis of cardiac rhabdomyoma
Authors: Gamzu Ronni , Achiron Reuven , Hegesh Julius , Weiner Ehud , Tepper Ron , Nir Amiram , Rabinowitz Ron , Auslander Ron , Yagel Simcha , Zalel Yaron , Zimmer Etan
Published: November 2002

Objective

To evaluate the prenatal parameters that increase the risk of tuberous sclerosis in prenatal management of fetal cardiac tumors suspected as rhabdomyoma.

Methods

The study was a retrospective survey of 18 documented cases in which cardiac rhabdomyoma was suspected during pregnancy. The following parameters were evaluated as possible risk factors associated with tuberous sclerosis: tumor size, isolated or multiple, and family history of tuberous sclerosis.

Results

Eighteen documented cases in which cardiac rhabdomyoma was found during pregnancy were evaluated. Of these cases, seven (39%) had proven tuberous sclerosis and 11 were found to be non‐associated tuberous sclerosis tumors. When combining the present data with previous series, cases with diagnosis of tuberous sclerosis had equal mean tumor size to those with normal outcome. Family history of tuberous sclerosis in the presence of cardiac rhabdomyoma almost invariably ended with tuberous sclerosis (86%). All other cases with diagnosis of tuberous sclerosis and no family history had all multiple cardiac tumors.

Conclusion

The present data suggest that 39% of in utero suspected cardiac rhabdomyoma would have tuberous sclerosis. Family history and multifocality remain the strongest predictors of tuberous sclerosis, whereas size of the cardiac tumor can not reliably be used to predict tuberous sclerosis in prenatal counseling. Copyright © 2002 John Wiley & Sons, Ltd.
Low or absent unconjugated estriol in pregnancy: an indicator for steroid sulfatase deficiency detectable by fluorescence in situ hybridization and biochemical analysis
Authors: Kashork Catherine D. , Sutton V. Reid , Fonda Allen Jill S. , Schmidt Deborah E. , Likhite Marisa L. , Potocki Lorraine , O'Brien William E. , Shaffer Lisa G.
Published: November 2002

It has been previously reported that a low or absent maternal serum unconjugated estriol (uE3) level is associated with placental steroid sulfatase (STS) deficiency. Here we report a correlation between patients who present with a very low or absent maternal serum uE3 and a deletion of the STS gene as assessed by fluorescence in situ hybridization (FISH). We studied nine prenatal cases that presented to the clinical laboratory with an abnormal triple screen, specifically low or absent maternal serum uE3 and a 46,XY karyotype. FISH analysis showed complete deletion of a probe containing the STS gene in six cases and one case had a partial deletion (reduced but not absent signal). The remaining two cases were not deleted for the STS probe. All mothers tested whose fetus showed a deletion were shown to be STS deletion carriers using FISH. Biochemical analysis was performed on 7/9 prenatal specimens. All fetuses deleted for the STS probe were also found to be deficient for STS by biochemical analysis of cultured amniotic fluid (5/5). Of the two fetuses not deleted for the STS probe, one was deficient for STS activity, while the other had a normal result. The abnormal result of enzyme deficiency by biochemical analysis in a non‐deletion case likely represents a mutation in the STS gene, not detectable by this FISH assay. Postnatal FISH confirmation of the STS deletion was performed in 1/7 cases. Clinical follow‐up was available for 4/9 cases following birth. Copyright © 2002 John Wiley & Sons, Ltd.
Oxygen induction of a novel fatty acid n−6 desaturase in the soil protozoon, Acanthamoeba castellanii
Authors: RUTTER Andrew J. , THOMAS Katie L. , HERBERT Derek , HENDERSON R. James , LLOYD David , HARWOOD John L.
Published: November 2002

Induction of fatty acid desaturation is very important for the temperature adaptation of poikilotherms. However, in oxygen-limited late-exponential-phase Acanthamoebacastellanii cultures, oxygen alone was able to induce increased activity of a fatty acid desaturase that converts oleate into linoleate and which has been implicated in the temperature adaptation of this organism. Experiments with Δ10-nonadecenoate showed that the enzyme is an n-6 desaturase rather than a Δ12-desaturase. It also used preferentially 1-acyl-2-oleoyl-phosphatidylcholine as substrate and NAD(P)H as electron donor. The involvement of cytochrome b5 as an intermediate electron carrier was shown by difference spectra measurements and anti-(cytochrome b5) antibody experiments. Of the three protein components of the desaturase complex, oxygen only increased the activity of the terminal (cyanide-sensitive) protein during n-6 desaturase induction. The induction of this terminal protein paralleled well the increase in overall oleate n-6 desaturation. The ability of oxygen to induce oleate desaturase independently of temperature in this lower eukaryotic animal model is of novel intrinsic interest, as well as being important for the design of future experiments to determine the molecular mechanism of temperature adaptation in poikilotherms.
Differential effects of reduced glycoprotein VI levels on activation of murine platelets by glycoprotein VI ligands
Authors: SNELL Daniel C. , SCHULTE Valerie , JARVIS Gavin E. , ARASE Kanako , SAKURAI Daiju , SAITO Takashi , WATSON Steve P. , NIESWANDT Bernhard
Published: November 2002

We have investigated the effects of decreased levels of the complex between glycoprotein VI (GPVI) and the Fc receptor γ-chain (FcRγ) on responses to collagen and GPVI-specific ligands in murine platelets. We show that levels of GPVI—FcRγ of the order of 50% and 20% of wild-type levels caused 2- and 5-fold shifts to the right respectively in the dose—response curve for aggregation in response to collagen, the snake toxin convulxin and the monoclonal antibody JAQ1. In addition, there is a delay in the onset of aggregation in response to collagen. In contrast, the stimulation of protein tyrosine phosphorylation by collagen (as measured after 150s) and adhesion to a collagen-coated surface under static conditions were unaffected in platelets with 50% and 20% of wild-type levels of GPVI. In contrast, responses to a collagen-related peptide (CRP), made up of repeat glycine-proline-hydroxyproline motifs, were markedly inhibited and abolished in platelets expressing 50% and 20% of wild-type levels of GPVI respectively. We suggest that the marked effect of a reduction in GPVI levels on the CRP-induced activation of platelets is due to the multivalent nature of CRP and the fact that GPVI is its sole receptor on platelets. Thus it appears that the interaction of CRP with GPVI is determined by a combination of affinity and avidity. The observation that collagen does not behave like CRP in platelets expressing reduced levels of GPVI, even in the combined presence of blocking antibodies against integrin α2β1 and GPV, suggests that collagen has a greater affinity than CRP for GPVI, and/or that other receptors are involved in its binding to platelets. The clinical significance of these results is discussed.
A potential role for nuclear factor of activated T-cells in receptor tyrosine kinase and G-protein-coupled receptor agonist-induced cell proliferation
Authors: YELLATURU Chandrahasa R. , GHOSH Salil K. , RAO R.K. , JENNINGS Lisa K. , HASSID Aviv , RAO Gadiparthi N.
Published: November 2002

We have studied the role of nuclear factor of activated T-cells (NFAT) transcription factors in the induction of vascular smooth muscle cell (VSMC) growth by platelet-derived growth factor-BB (PDGF-BB) and thrombin, the receptor tyrosine kinase (RTK) and G-protein-coupled receptor (GPCR) agonists, respectively. NFATc1 but not NFATc2 or NFATc3 was translocated from the cytoplasm to the nucleus upon treatment of VSMCs with PDGF-BB or thrombin. Translocation of NFATc1 was followed by an increase in NFAT—DNA binding activity and NFAT-dependent reporter gene expression. Cyclosporin A (CsA), a potent and specific inhibitor of calcineurin, a calcium/calmodulin-dependent serine phosphatase involved in the dephosphorylation and activation of NFATs, blocked NFAT—DNA binding activity and NFAT-dependent reporter gene expression induced by PDGF-BB and thrombin. CsA also completely inhibited PDGF-BB- and thrombin-induced VSMC growth, as measured by DNA synthesis and cell number. In addition, forced expression of the NFAT-competing peptide VIVIT for calcineurin binding significantly attenuated the DNA synthesis induced by PDGF-BB and thrombin in VSMCs. Together, these findings for the first time demonstrate a role for NFATs in RTK and GPCR agonist-induced growth in VSMCs.
Ca2+ promotes erythrocyte band 3 tyrosine phosphorylation via dissociation of phosphotyrosine phosphatase from band 3
Authors: ZIPSER Yehudit , PIADE Adi , BARBUL Alexander , KORENSTEIN Rafi , KOSOWER Nechama S.
Published: November 2002

The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by protein tyrosine kinase (PTK). We have previously identified a band 3-associated phosphotyrosine phosphatase (PTP) that is normally highly active and prevents the accumulation of band 3 phosphotyrosine. Band 3 tyrosine phosphorylation can be induced by inhibition of PTP (vanadate, thiol oxidation), activation of PTK (hypertonic NaCl) or intracellular increased Ca2+ (mechanism unknown). We now show that there is inhibition of dephosphorylation of band 3 in Ca2+/ionophore-treated erythrocytes and in membranes isolated from the treated cells. These membranes exhibit phosphatase activity upon the addition of exogenous substrate. Dephosphorylation of the endogenous substrate (band 3) can be activated in these membranes by the addition of Mg2+. Thus the inability of PTP to dephosphorylate the band 3 phosphotyrosine is not due to inhibition of the enzyme itself. Ca2+ rise in the erythrocyte causes dissociation of PTP from band 3, thus leaving the kinase unopposed. This is shown by a significant diminution in band 3/PTP co-precipitation. Addition of Mg2+ to these membranes leads to reassociation of band 3 with PTP. The Ca2+-induced inhibition of band 3 dephosphorylation may be due to Ca2+-dependent alterations in membrane components and structure, affecting the interaction of band 3 with PTP. The Ca2+-induced tyrosine phosphorylation, involving an apparent PTP inhibition via dissociation from the substrate, may play a role in signal transduction pathways and in certain pathological disorders associated with increased cell Ca2+.
Role of proline, cysteine and a disulphide bridge in the structure and activity of the anti-microbial peptide gaegurin 5
Authors: PARK Sang-Ho , KIM Hyung-Eun , KIM Chi-Man , YUN Hee-Jeong , CHOI Eung-Chil , LEE Bong-Jin
Published: November 2002

Gaegurin 5 (GGN5) is a cationic 24-residue anti-microbial peptide isolated from the skin of a Korean frog, Rana rugosa. It contains a central proline residue and an intra-residue disulphide bridge in its C-terminus, which are common to the anti-microbial peptides found in Ranidae. We determined the solution structure of GGN5 bound to SDS micelles for the first time and investigated the role of proline, cysteine and a disulphide bridge on the structure and activity of GGN5. GGN5 adopts an amphipathic α-helical structure spanning residues 3—20 kinked around Pro-14, which allows the hydrophobic residues to reside in the concave helical region, and a disulphide-bridged loop-like conformation in its C-terminus. By replacement of proline with alanine (PAGGN5), a straight and rigid helix was formed in the central region and was more stable than the kinked helix. Reduction of a disulphide bridge in the C-terminus (GGN5SH) maintained the loosely ordered loop-like conformation, while the replacement of two cysteines with serines (CSGGN5) caused the C-terminal conformation to be completely disordered. The magnitude of anti-microbial activity of the peptides was closely related to their helical stability in the order PAGGN5>GGN5>GGN5SH>CSGGN5, suggesting that the helical stability of the peptides is important for anti-microbial activity. On the other hand, the significant increase of haemolytic activity of PAGGN5 implies that a helical kink of GGN5 could be involved in the selectivity of target cells. The location of GGN5 and PAGGN5, analysed using paramagnetic probes, was mainly at the surface of SDS micelles, although the location of the N-terminal region was slightly different between them.
Functional characterization and structural modelling of late gene expression factor 4 from Bombyx mori nucleopolyhedrovirus
Authors: SEHRAWAT Seema , SRINIVASAN Narayanaswamy , GOPINATHAN Karumathil P.
Published: November 2002

Late gene expression factor 4 (LEF4), a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus has been bacterially expressed and characterized. Sequence analyses and three-dimensional modelling of B. mori LEF4 showed that the protein is related to mRNA-capping enzymes, which are organized as two modular domains. Most of the acidic side chains in LEF4 were solvent-exposed and spread all along the fold. A region dominated by negatively charged groups, which protrudes from the larger domain was ideally suited for interactions with proteins having positively charged patches at the surface. The purified LEF4 protein exhibited different enzyme activities associated with mRNA-capping enzymes, i.e. GTP-binding, RNA triphosphatase and guanylate transferase activities. In addition, LEF4 also showed NTP-hydrolysing activity. The kinetic analysis of ATP hydrolysis revealed a sigmoidal response with two deduced binding sites for ATP, whereas the guanylate transferase activity showed a typical hyperbolic response to varying concentrations of GTP with a Km of 330±20μM. Analysis of the modelled three-dimensional structure of LEF4 suggested the presence of crucial residues in sequence motifs important for the integrity of the fold. Mutation of one such conserved and buried tyrosine residue to cysteine in the motif IIIa, located close to the interlobe region of the model, resulted in a 44% loss of guanylate transferase activity of LEF4 but had no effect on the ATPase activity.
Mitogen-activated protein kinases and nuclear factor-κB regulate Helicobacter pylori-mediated interleukin-8 release from macrophages
Authors: BHATTACHARYYA Asima , PATHAK Shresh , DATTA Simanti , CHATTOPADHYAY Santanu , BASU Joyoti , KUNDU Manikuntala
Published: November 2002

Gastric infection, as well as inflammation, caused by Helicobacter pylori, activates the production of cytokines and chemokines by mononuclear cells; interleukin-8 (IL-8) is one of the major inflammatory chemokines. Since H. pylori does not invade mucosal tissue, we observed the effect of the water extract of H. pylori (HPE), containing shed factors, on the production of IL-8 by human peripheral blood monocytes and the human monocyte cell line THP-1. HPE-treatment induced activation of the mitogen-activated protein kinases (MAPKs) ERK (extracellular signal-regulated kinase), p38 and JNK (c-Jun N-terminal kinase), an effect which was not dependent on the presence of the cag pathogenicity island. p38 MAPK activation was sustained. The specific inhibitors, U0126 (for ERK1/2 signalling) and SB203580 (for p38 MAPK signalling), both abrogated IL-8 secretion from HPE-treated THP-1. Dominant-negative mutants of the upstream kinases MEK1 (MAPK/ERK kinase 1), MKK (MAPK kinase) 6 and MKK7 also inhibited IL-8 secretion, pointing to a role of all three MAPKs in HPE-mediated IL-8 release. The inhibitory effects of polymyxin B and anti-CD14 antibody suggested that the effect of HPE on MAPKs was mediated by H. pylori lipopolysaccharide (LPS). By analysis of IL-8-promoter-driven luciferase gene expression, we observed that the effects of HPE-induced nuclear factor-κB (NF-κB) activation and MAPK signalling were mediated at the level of the IL-8 promoter. While ERK1/2 activation could be linked to enhanced DNA binding of activator protein-1 (AP-1), p38 MAPK signalling did not affect AP-1 DNA binding. Taken together, these results provide the first evidence that LPS from H. pylori stimulates IL-8 release from cells of the monocytic lineage through activation of NF-κB and signalling along MAPK cascades. The stimulation of MAPK signalling in macrophages by LPS of H. pylori amplifies the inflammatory response associated with gastric H. pylori infection and needs to be taken into consideration when developing therapeutics based on these signalling pathways.
Endothelial nitric oxide synthase (NOS) deficiency affects energy metabolism pattern in murine oxidative skeletal muscle
Authors: MOMKEN Iman , FORTIN Dominique , SERRURIER Bernard , BIGARD Xavier , VENTURA-CLAPIER Renée , VEKSLER Vladimir
Published: November 2002

Oxidative capacity of muscles correlates with capillary density and with microcirculation, which in turn depend on various regulatory factors, including NO generated by endothelial nitric oxide synthase (eNOS). To determine the role of eNOS in patterns of regulation of energy metabolism in various muscles, we studied mitochondrial respiration in situ in saponin-permeabilized fibres as well as the energy metabolism enzyme profile in the cardiac, soleus (oxidative) and gastrocnemius (glycolytic) muscles isolated from mice lacking eNOS (eNOS-/-). In soleus muscle, the absence of eNOS induced a marked decrease in both basal mitochondrial respiration without ADP (-32%; P<0.05) and maximal respiration in the presence of ADP (-29%; P<0.05). Furthermore, the eNOS-/- soleus muscle showed a decrease in total creatine kinase (-29%; P<0.05), citrate synthase (-31%; P<0.01), adenylate kinase (-27%; P<0.05), glyceraldehyde-3-phosphate dehydrogenase (-43%; P<0.01) and pyruvate kinase (-26%; P<0.05) activities. The percentage of myosin heavy chains I (slow isoform) was significantly increased from 24.3±1.5% in control to 30.1±1.1% in eNOS-/- soleus muscle (P<0.05) at the expense of a slight non-significant decrease in the three other (fast) isoforms. Besides, eNOS-/- soleus showed a 28% loss of weight. Interestingly, we did not find differences in any parameters in cardiac and gastrocnemius muscles compared with respective controls. These results show that eNOS knockout has an important effect on muscle oxidative capacity as well on the activities of energy metabolism enzymes in oxidative (soleus) muscle. The absence of such effects in cardiac and glycolytic (gastrocnemius) muscle suggests a specific role for eNOS-produced NO in oxidative skeletal muscle.
An E2F-binding site mediates the activation of the proliferative isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by phosphatidylinositol 3-kinase
Authors: FERNÁNDEZ de MATTOS Silvia , LAM Eric W.-F. , TAULER Albert
Published: November 2002

In the present study, we demonstrate that E2F is implicated in the regulation of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) during cell division. The expression of this enzyme is induced during the G1/S transition of the cell cycle. We identified and monitored the E2F-pocket protein complexes that bind to the E2F site of the F-type promoter during cell-cycle entry, and we analysed their contribution to the phosphatidylinositol 3-kinase (PI 3-kinase)-mediated regulation of the promoter. We found that the predominant E2F complex bound to the F-type promoter in unstimulated/quiescent cells contains E2F4, DP1 and p130 proteins. In serum-stimulated (S-phase) cells, the composition of the complex switched to E2F1/4, DP1 and p107, together with cyclin A and cyclin-dependent kinase 2. Treatment with the PI 3-kinase specific inhibitor LY 294002 prevented the formation of the S-phase complex, suggesting that activation of the PI 3-kinase pathway is essential for the formation of this complex. Further supporting this idea, we obtained results showing that treatment of cycling NIH 3T3 cells with either wortmannin or LY 294002 induces the accumulation of the transcriptionally repressive p130—E2F4—DP1 complex. Using the Rat-1 ER—E2F1 cell line where E2F1 activity can be conditionally induced, we demonstrated that E2F activity is involved in the in vivo transcriptional regulation of the F-type 6PF2K/Fru-2,6-BPase gene. Taken together, our results show that the F-type 6PF2K/Fru-2,6-BPase is a genuine E2F-regulated gene, and that its regulation by the PI 3-kinase pathway is at least partially mediated through the E2F transcription factor.
Transcriptional regulation of the human glycoprotein hormone common α subunit gene by cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and p53
Authors: ZHANG Xian , GRAND Roger J.A. , McCABE Christopher J. , FRANKLYN Jayne A. , GALLIMORE Phillip H. , TURNELL Andrew S.
Published: November 2002

We have investigated the functional interactions between adenovirus early region 1A (AdE1A) protein, the co-activators cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and SUG1, and the transcriptional repressor retinoblastoma (Rb) in mediating T3-dependent repression. Utilizing the human glycoprotein hormone common α-subunit (α-subunit) promoter and AdE1A mutants with selective binding capacity to these molecules we have determined an essential role for CBP/p300. In normal circumstances, wild-type 12S AdE1A inhibited α-subunit activity. In contrast, adenovirus mutants that retain both the SUG1- and Rb-binding sites, but lack the CBP/p300-binding site, were unable to repress promoter activity. We have also identified a role for the tumour-suppressor gene product p53 in regulation of the α-subunit promoter. Akin to 12S AdE1A, exogenous p53 expression repressed α-subunit activity. This function resided in the ability of p53 to interact with CBP/p300; an N-terminal mutant incapable of interacting with CBP/p300 did not inhibit α-subunit activity. Stabilization of endogenous p53 by UV irradiation also correlated positively with reduced α-subunit activity. Intriguingly, T3 stimulated endogenous p53 transcriptional activity, implicating p53 in T3-dependent signalling pathways. These data indicate that CBP/p300 and p53 are key regulators of α-subunit activity.
The genes pme-1 and pme-2 encode two poly(ADP-ribose) polymerases in Caenorhabditis elegans
Authors: GAGNON Steve N. , HENGARTNER Michael O. , DESNOYERS Serge
Published: November 2002

Poly(ADP-ribose) polymerases (PARPs) are an expanding, well-conserved family of enzymes found in many metazoan species, including plants. The enzyme catalyses poly(ADP-ribosyl)ation, a post-translational modification that is important in DNA repair and programmed cell death. In the present study, we report the finding of an endogenous source of poly(ADP-ribosyl)ation in total extracts of the nematode Caenorhabditis elegans. Two cDNAs encoding highly similar proteins to human PARP-1 (huPARP-1) and huPARP-2 are described, and we propose to name the corresponding enzymes poly(ADP-ribose) metabolism enzyme 1 (PME-1) and PME-2 respectively. PME-1 (108kDa) shares 31% identity with huPARP-1 and has an overall structure similar to other PARP-1 subfamily members. It contains sequences having considerable similarity to zinc-finger motifs I and II, as well as with the catalytic domain of huPARP-1. PME-2 (61kDa) has structural similarities with the catalytic domain of PARPs in general and shares 24% identity with huPARP-2. Recombinant PME-1 and PME-2 display PARP activity, which may partially account for the similar activity found in the worm. A partial duplication of the pme-1 gene with pseudogene-like features was found in the nematode genome. Messenger RNA for pme-1 are 5′-tagged with splice leader 1, whereas those for pme-2 are tagged with splice leader 2, suggesting an operon-like expression for pme-2. The expression pattern of pme-1 and pme-2 is also developmentally regulated. Together, these results show that PARP-1 and −2 are conserved in evolution and must have important functions in multicellular organisms. We propose using C. elegans as a model to understand better the functions of these enzymes.
Oligolysine-based saccharide clusters: synthesis and specificity
Authors: FRISON Natacha , MARCEAU Philippe , ROCHE Annie-Claude , MONSIGNY Michel , MAYER Roger
Published: November 2002

In search of specific and highly selective sugar clusters for cell receptors, such as membrane lectins, various disaccharides were coupled to small peptide cores through an amide bond. In a first step, the reducing disaccharides, i.e. lactose and three different dimannoses, were converted into glycosyl-pyroglutamyl-β-alanine derivatives. The free carboxylic group of these conjugates was then coupled to the α and ∊ amino groups of the core peptide (Lysn-Ala-Cys-NH2) with n = 1 to 5, with complete substitution leading to homogeneous glycoclusters. The thiol group of the cysteine residue was used to tag the glycosylated oligolysines upon reaction with fluorescein iodoacetamide. The affinity of these glycoclusters towards two plant lectins was assessed by surface plasmon resonance. The selectivity of their cell uptake was investigated by flow cytometry using two types of cells: a human hepatoma cell line (HepG2 cells) expressing the plasma membrane galactose-specific lectin, and monocyte-derived dendritic cells expressing the plasma membrane mannose-specific lectin. The glycoclusters containing four or five disaccharides were shown to bind plant lectins and cell surface membrane lectins with a narrow selectivity and with a high affinity.
Phosphorylation is required for PMA- and cell-cycle-induced degradation of protein kinase Cδ
Authors: SRIVASTAVA Jyoti , PROCYK Katarzyna J. , ITURRIOZ Xavier , PARKER Peter J.
Published: November 2002

Classical and novel protein kinase C (PKC) isoforms are down-regulated as a result of chronic activation by certain tumour promoters and physiological stimuli; however, the mechanisms leading to down-regulation are not fully understood. In the present study, we have studied the PMA ('TPA')-induced degradation of PKCΔ in NIH 3T3 cells under culture conditions where PKCΔ displays cell-cycle-dependent down-regulation. In contrast with previous studies, a hyperphosphorylated form of this PKC isoform, promoted by calyculin A, was rapidly degraded in PMA-treated cells. Similarly, the presence of calyculin A enhanced the down-regulation of PKCΔ observed on G1/S-phase progression through the cell cycle. Analysis of phosphorylation-site mutants indicated that the T-loop Thr505 phosphorylation site was critical for induced degradation.
Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites
Authors: SINGH Ajay , SHENAI Bhaskar R. , CHOE Youngchool , GUT Jiri , SIJWALI Puran S. , CRAIK Charles S. , ROSENTHAL Philip J.
Published: November 2002

Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity (kcat/Km increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.
Cell-surface prion protein interacts with glycosaminoglycans
Authors: PAN Tao , WONG Boon-Seng , LIU Tong , LI Ruliang , PETERSEN Robert B. , SY Man-Sun
Published: November 2002

We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23—35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs.
Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles
Authors: RINALDI Andrea C. , MANGONI Maria Luisa , RUFO Anna , LUZI Carla , BARRA Donatella , ZHAO Hongxia , KINNUNEN Paavo K.J. , BOZZI Argante , GIULIO Antonio DI , SIMMACO Maurizio
Published: November 2002

The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings.
Evidence for crucial electrostatic interactions between Bcl-2 homology domains BH3 and BH4 in the anti-apoptotic Nr-13 protein
Authors: LALLE Philippe , AOUACHERIA Abdel , DUMONT-MISCOPEIN Agnès , JAMBON Martin , VENET Séverine , BOBICHON Hélène , COLAS Pierre , DELÉAGE Gilbert , GEOURJON Christophe , GILLET Germain
Published: November 2002

Nr-13 is an anti-apoptotic member of the Bcl-2 family previously shown to interact with Bax. The biological significance of this interaction was explored both in yeast and vertebrate cells and revealed that Nr-13 is able to counteract the pro-apoptotic activity of Bax. The Bax-interacting domain has been identified and corresponds to α-helices 5 and 6 in Nr-13. Site-directed mutagenesis has revealed that the N-terminal region of Nr-13 is essential for activity and corresponds to a genuine Bcl-2 homology domain (BH4). The modelling of Nr-13, based on its similarity with other Bcl-2 family proteins and energy minimization, suggests the possibility of electrostatic interactions between the two N-terminal-conserved domains BH4 and BH3. Disruption of these interactions severely affects Nr-13 anti-apoptotic activity. Together our results suggest that electrostatic interactions between BH4 and BH3 domains play a role in the control of activity of Nr-13 and a subset of Bcl-2 family members.
Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides
Authors: CEZARI Maria Helena S. , PUZER Luciano , JULIANO Maria Aparecida , CARMONA Adriana K. , JULIANO Luiz
Published: November 2002

We have examined in detail the specificity of the subsites S1, S2, S1′ and S2′ for the carboxydipeptidase activity of cathepsin B by synthesizing and assaying four series of internally quenched fluorescent peptides based on the sequence Dnp-GFRFW-OH, where Dnp (2,4-dinitrophenyl) is the quenching group of the fluorescence of the tryptophan residue. Each position, except the glycine, was substituted with 15 different naturally occurring amino acids. Based on the results we obtained, we also synthesized efficient and sensitive substrates that contained o-aminobenzoic acid and 3-Dnp-(2,3-diaminopropionic acid), or ∊-amino-Dnp-Lys, as the fluorescence donor—receptor pair. The higher kinetic parameter values for the carboxydipeptidase compared with the endopeptidase activity of cathepsin B allowed an accurate analysis of its specificity. The subsite S1 accepted preferentially basic amino acids for hydrolysis; however, substrates with phenylalanine and aliphatic side-chain-containing amino acids at P1 had lower Km values. Despite the presence of Glu245 at S2, this subsite presented clear preference for aromatic amino acid residues, and the substrate with a lysine residue at P2 was hydrolysed better than that containing an arginine residue. S1′ is essentially a hydrophobic subsite, and S2′ has particular preference for phenylalanine or tryptophan residues.
Syntaxin 7, syntaxin 8, Vti1 and VAMP7 (vesicle-associated membrane protein 7) form an active SNARE complex for early macropinocytic compartment fusion in Dictyostelium discoideum
Authors: BOGDANOVIC Aleksandra , BENNETT Nelly , KIEFFER Sylvie , LOUWAGIE Mathilde , MORIO Takahiro , GARIN Jérôme , SATRE Michel , BRUCKERT Franz
Published: November 2002

The macropinocytic pathway in Dictyostelium discoideum is organized linearly. After actin-driven internalization, fluid material passes sequentially from endosomes to lysosomes, where molecules are degraded and absorbed. Residual material is exocytosed via post-lysosomal compartments. Syntaxin 7 is a SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein that is present and active in D. discoideum endosomes [Bogdanovic, Bruckert, Morio and Satre (2000) J. Biol. Chem. 275, 36691—36697]. Here we report the identification of its main SNARE partners by co-immunoprecipitation and MS peptide sequencing. The syntaxin 7 complex contains two co-t-SNAREs [Vti1 (Vps10p tail interactor 1) and syntaxin 8] and a v-SNARE [VAMP7 (vesicle-associated membrane protein 7)] (where t-SNAREs are SNAREs of the target compartment and v-SNAREs are SNAREs present in donor vesicles). In endosomes and in vitro, syntaxin 7, Vti1 and syntaxin 8 form a complex that is able to bind VAMP7. Antibodies to syntaxin 8 and a soluble recombinant VAMP7 fragment both inhibit in vitro reconstituted D. discoideum endosome fusion. The lysosomal content of syntaxin 7, Vti1, syntaxin 8 and VAMP7 is low compared with that in endosomes, implying a highly active recycling or retention mechanism. A likely model is that VAMP7 is a v-SNARE present on vesicles carrying lysosomal enzymes, and that the syntaxin 7—Vti1—syntaxin 8 t-SNARE complex is associated with incoming endocytic material.
Synthesis and secretion of Muc4/sialomucin complex: implication of intracellular proteolysis
Authors: KOMATSU Masanobu , ARANGO Maria E. , CARRAWAY Kermit L.
Published: November 2002

Muc4/sialomucin complex (SMC) is a heterodimeric glycoprotein complex implicated in epithelial protection and overexpressed in some tumours. It is encoded by a single gene, and the two subunits are produced by proteolytic cleavage at a time before substantial O-glycosylation, near the time of transit from the endoplasmic reticulum to the Golgi. Although Muc4/SMC is translated as a membrane protein, it is produced as a soluble form in many epithelia. Transfection experiments using Cos-7, HBL-100 human epithelial, MCF-7 human breast tumour and HC11 mouse mammary cell lines showed that soluble rat Muc4/SMC is produced by a proteolytic cleavage mechanism and released by secretion from multiple cell lines, including both human and mouse normal epithelial cells and tumour cells. Similar transfection experiments demonstrated the same mechanism for the human analogue MUC4. Gel electrophoresis analyses of deglycosylated membrane and soluble transmembrane subunits and of the membrane-associated cleavage fragment indicated a released cleavage product of 25kDa, resulting from cleavage between two epidermal growth factor-like domains. Further evidence for this site was obtained from deletion mutants removing this region of the protein, which blocked secretion. Finally, pulse—chase analyses of Muc4/SMC biosynthesis indicated no kinetic difference between the timing of the cleavage to release the soluble form and that to produce the two subunits, indicating that the soluble form is created early in transit to the cell surface. These studies provide the first clear evidence that membrane mucins can be released from cells by an intracellular proteolytic mechanism that leads to secretion of the soluble form of the mucin.
A locus on human chromosome 20 contains several genes expressing protease inhibitor domains with homology to whey acidic protein
Authors: CLAUSS Adam , LILJA Hans , LUNDWALL Åke
Published: November 2002

A locus containing 14 genes, encoding protein domains that have homology with whey acidic protein (WAP), has been identified in a region of 678kb on human chromosome 20q12-13.1. Among them are genes of the known or postulated protease inhibitors elafin, secretory leucocyte protease inhibitor, human epididymis gene product 4, eppin, and huWAP2. Nucleotide sequences of full-length transcripts were obtained from cDNA fragments generated by rapid amplification of cDNA ends. Characteristic features of the genes are that the upstream promoter regions are devoid of TATA-boxes and that the coding nucleotides are divided into distinct exons for the signal peptide and for each WAP domain. In most cases, there is also a separate exon encompassing a few terminal codons and the 3′ untranslated nucleotides. There are also examples of mixed type inhibitors, that encode inhibitor domains of both WAP and Kunitz types. Several of the genes appear to be expressed ubiquitously, but, in most cases, the highest transcript levels are found in epididymis followed by testis and trachea. Some of the genes also display high transcript levels in neural tissues. Potential biological roles of protein products could be in host defence against invading micro-organisms or in the regulation of endogenous proteolytic enzymes, of which those originating from the kallikrein gene locus on chromosome 19 are of particular interest.
Phosphorylation of SNAP-25 on serine-187 is induced by secretagogues in insulin-secreting cells, but is not correlated with insulin secretion
Authors: GONELLE-GISPERT Carmen , COSTA Maria , TAKAHASHI Masami , SADOUL Karin , HALBAN Philippe
Published: November 2002

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25kDa (SNAP-25) is implicated in regulated insulin secretion. In pheochromocytoma PC12 cells, SNAP-25 is phosphorylated at Ser187, which lies in a region that is important for its function. The aims of the present study were to determine whether SNAP-25 is phosphorylated at Ser187 in insulin-secreting cells and, if so, whether this is important for regulated insulin secretion. The major findings are: (i) SNAP-25 is rapidly and reversibly phosphorylated on Ser187 in both rat insulinoma INS-1 cells and rat islets in response to the phorbol ester, PMA; (ii) less than 35% of SNAP-25 in INS-1 cells is phosphorylated in response to PMA, and phosphorylation is limited to plasma-membrane-associated SNAP-25; (iii) both SNAP-25 isoforms (a and b) are phosphorylated, with 1.8-fold greater phosphorylation for SNAP-25b in response to PMA; (iv) in rat islets, Ser187 phosphorylation is stimulated by glucose or carbachol, albeit to a lesser extent than by PMA, but not by cAMP; (v) insulin secretion from botulinum neurotoxin E-treated hamster insulinoma tumour (HIT) cells, transfected with toxin-resistant Ser187→Ala or Ser187→Asp mutant SNAP-25, was similar to that of wild-type HIT cells. Furthermore, in rat islets no correlation was found between the extent of SNAP-25 phosphorylation at Ser187 in response to secretagogues and stimulation of insulin release; (vi) use of protein kinase C (PKC) inhibitors suggests that glucose stimulates SNAP-25 phosphorylation via conventional and non-conventional PKC isoforms. In summary, although SNAP-25 phosphorylation at Ser187 occurs in insulin-secreting cells and is mediated by PKC, it does not appear to play a major role in regulated insulin secretion.
N-glycosylation is required for full enzymic activity of the murine galactosylceramide sulphotransferase
Authors: ECKHARDT Matthias , FEWOU Simon N. , ACKERMANN Ivonne , GIESELMANN Volkmar
Published: November 2002

3-O-Sulphogalactosylceramide (sulphatide) is a major lipid component of myelin membranes, and is required for proper myelin formation. Sulphatide is synthesized in the Golgi apparatus by galactosylceramide sulphotransferase (CST; EC 2.8.2.11). Murine and human CSTs contain two putative N-glycosylation sites (Asn-66 and Asn-312). The second site is conserved among all galactose 3-O-sulphotransferases cloned to date. In order to study the functional relevance of N-glycosylation, we generated epitope-tagged CST and soluble Protein A—CST fusion proteins lacking both N-glycosylation sites, separately or in combination. Our results show that both sites are glycosylated when CST is expressed in Chinese hamster ovary (CHO) or COS cells. Moreover, transfecting CST mutants lacking both N-glycosylation sites, or only Asn-312, reduced significantly the amount of sulphatide synthesized, whereas substituting Asn-66 with a glutamine residue did not. In contrast, activity in vitro was reduced by approx. 50% in the Asn-66→Gln (N66Q) mutant, and was almost undetectable in N312Q and N66/312Q transfectants. Furthermore, soluble Protein A—CST expressed in the presence of tunicamycin was almost inactive, and accumulated in transfected cells. Expression of fully active CST in a CHO-glycosylation mutant lacking N-acetylglucosaminyltransferase I demonstrated that condensation of the N-linked pentamannosyl-core structure is sufficient to form a fully active enzyme.
Identification of a plasma membrane glutamine transporter from the rat hepatoma cell line H4-IIE-C3
Authors: POLLARD Matthew , MEREDITH David , McGIVAN John D.
Published: November 2002

Glutamine is taken up into the rat hepatoma cell line H4-IIE-C3 by a Na+-dependent transport system which is specific for glutamine, alanine, serine, cysteine and asparagine and does not tolerate substitution of Na+ by Li+. Glutamine transport was relatively weakly inhibited by a 50-fold excess of leucine and was not inhibited by phenylalanine or N-methyl aminoisobutyrate. These general properties are characteristic of the recently identified ASCT/B0 family of transporters. Using a reverse transcriptase PCR-based homology cloning approach, we have characterized a cDNA for a novel member of this transporter family (H4-ASCT2) from H4-IIE-C3 cells. The cDNA encodes a 551-amino acid protein which exhibits similarities of between 75 and 85% with ASCT/B0 transporters previously cloned from other sources. When expressed in Xenopus oocytes, this transporter catalyses Na+-dependent glutamine uptake with characteristics very similar to those of glutamine uptake into the H4-IIE-C3 cells. This newly characterized transporter possesses a number of amino acid sequence differences from ASCT2 clones recently isolated from rat astroglial cells and from normal rat liver. In particular, the loop region between transmembrane helices 3 and 4 from H4-ASCT2 shares less than 60% sequence similarity with ASCT2 from rat liver; furthermore, there are some 25 single amino acid substitutions elsewhere in the H4-ASCT2 sequence compared with that from rat liver. Thus enhanced glutamine uptake in rat hepatoma cells is mediated by the expression of a novel ASCT/B0 transporter isoform rather than by increased expression of the ASCT2 mRNA found in normal rat liver.
An intracellular protease of the crenarchaeon Sulfolobus solfataricus, which has sequence similarity to eukaryotic peptidases of the CD clan
Authors: GUAGLIARDI Annamaria , CERCHIA Laura , ROSSI Mosè
Published: November 2002

We purified from crude extracts of the hyperthermophilic crenarchaeon Sulfolobus solfataricus a protease that is able to hydrolyse proteins with a pH optimum of 7.5 and a temperature optimum of 70°C. Assays in the presence of classical protease inhibitors showed that the hydrolytic activity is sensitive to thiol-blocking reagents. Fluorescence assays using synthetic peptides demonstrated that the protease has a preference for cleaving glutamic acid residues. The first 12 residues of the protease match the N-terminus residues of a hypothetical protein in the S. solfataricus genome of 95 amino acids in length and calculated molecular mass of 11072Da. The whole sequence of the protease is not related to any known protein, but it bears a segment which is highly similar to one containing the active cysteine residue in eukaryotic peptidases known as legumains. This is the first protease isolated from S. solfataricus capable of degrading native proteins effectively. Our results add to the knowledge of the intracellular proteolytic machine in hyperthermophilic micro-organisms.
The cyclic dipeptide CI-4 [cyclo-(l-Arg-d-Pro)] inhibits family 18 chitinases by structural mimicry of a reaction intermediate
Authors: HOUSTON Douglas R. , EGGLESTON Ian , SYNSTAD Bj⊘rnar , EIJSINK Vincent G.H. , AALTEN Daan M.F. van
Published: November 2002

Family 18 chitinases are attractive targets for the development of new inhibitors with chemotherapeutic potential against fungi, insects and protozoan/nematodal parasites. Although several inhibitors have been identified, these are based on complex chemistry, which hampers iterative structure-based optimization. Here we report the details of chitinase inhibition by the natural product peptide CI-4 [cyclo-(l-Arg-d-Pro)], which possesses activity against the human pathogenic fungus Candida albicans, and describe a 1.7Å (0.17nm) crystal structure of CI-4 in complex with the enzyme. The structure reveals that the cyclic dipeptide inhibits chitinases by structurally mimicking a reaction intermediate, and could, on the basis of its accessible chemistry, be a candidate for further optimization.