bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing cathepsin K activity with a selective substrate spanning its active site
Authors: LECAILLE Fabien , WEIDAUER Enrico , JULIANO Maria A. , BRÖMME Dieter , LALMANACH Gilles
Published: October 2003

The limited availability of highly selective cathepsin substrates seriously impairs studies designed to monitor individual cathepsin activities in biological samples. Among mammalian cysteine proteases, cathepsin K has a unique preference for a proline residue at P2, the primary determinant of its substrate specificity. Interestingly, congopain from Trypanosoma congolense also accommodates a proline residue in its S2 subsite. Analysis of a congopain model showed that amino acids forming its S2 subsite are identical with those of cathepsin K, except Leu67 which is replaced by a tyrosine residue in cathepsin K. Furthermore, amino acid residues of the congopain S2′ binding pocket, which accepts a proline residue, are strictly identical with those of cathepsin K. Abz-HPGGPQ-EDN2ph [where Abz represents o-aminobenzoic acid and EDN2ph (=EDDnp) represents N-(2,4-dinitrophenyl)-ethylenediamine], a substrate initially developed for trypanosomal enzymes, was efficiently cleaved at the Gly–Gly bond by cathepsin K (kcat/Km=426000 M−1·s−1). On the other hand, Abz-HPGGPQ-EDN2ph was resistant to hydrolysis by cathepsins B, F, H, L, S and V (20 nM enzyme concentration) and the Y67L (Tyr67→Leu)/L205A cathepsin K mutant (20 nM), but still acted as a competitive inhibitor. Taken together, the selectivity of Abz-HPGGPQ-EDN2ph to cathepsin K primarily depends on the S2 and S2′ subsite specificities of cathepsin K and the ionization state of histidine at P3. Whereas Abz-HPGGPQ-EDN2ph was hydrolysed by wild-type mouse fibroblast lysates, its hydrolysis was completely abolished in the cathepsin K-deficient samples, indicating that Abz-HPGGPQ-EDN2ph can be used to monitor selectively cathepsin K activity in physiological fluids and cell lysates.
Induction of transcripts derived from promoter III of the acetyl-CoA carboxylase-α gene in mammary gland is associated with recruitment of SREBP-1 to a region of the proximal promoter defined by a DNase I hypersensitive site
Authors: BARBER Michael C. , VALLANCE Amanda J. , KENNEDY Helen T. , TRAVERS Maureen T.
Published: October 2003

ACC-α (acetyl-CoA carboxylase-α), a key regulator of fatty-acid metabolism, is encoded by mRNAs transcribed from three promoters, PI, PII and PIII, in the ovine genome. Enhanced expression of transcripts encoded by PIII in mammary gland during lactation is associated with alterations in chromatin structure that result in the detection of two DNase I hypersensitive sites, upstream of the start site. The most proximal site, located between −190 and −10, is characterized by the presence of an inverted-CCAAT box, C2 at −167, and E-boxes, E1 and E2, at −151 and −46. Deletion of these motifs, which bind nuclear factor-Y and upstream stimulatory factors respectively in gel-shift assays, attenuates the activity of luciferase reporter constructs in transfected cells. Chromatin immunoprecipitation demonstrated that these transcription factors were associated with PIII in vivo in both lactating and non-lactating mammary tissues. The basic helix–loop–helix-leucine zipper transcription factor, SREBP-1 (sterol-regulated-element-binding protein-1), transactivated PIII reporter constructs in transfected HC11 mammary cells, and this was dependent on the presence of E1, but not on C2 or E2. SREBP-1 was only associated with PIII in chromatin from lactating animals, which was coincident with a 4-fold increase in the precursor (125 kDa) form of SREBP-1 in microsomes and the appearance of the mature form (68 kDa) in the nucleus. SREBP-1 motifs are also present in the proximal region of PII, which is also induced in lactation. This indicates that SREBP-1 is a major developmental regulator of the programme of lipid synthesis de novo in the lactating mammary gland.
Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-α
Authors: FONTES Marcos R. M. , TEH Trazel , TOTH Gabor , JOHN Anna , PAVO Imre , JANS David A. , KOBE Bostjan
Published: October 2003

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-α (Impα), we co-crystallized non-autoinhibited Impα with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impα. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impα. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impα, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impα.
Regulation of type II transforming-growth-factor-β receptors by protein kinase C ι
Authors: CHUANG Lea-Yea , GUH Jinn-Yuh , LIU Shu-Fen , HUNG Min-Yuan , LIAO Tung-Nan , CHIANG Tai-An , HUANG Jau-Shyang , HUANG Yu-Lun , LIN Chi-Fong , YANG Yu-Lin
Published: October 2003

TGF-β (transforming growth factor-β) is implicated in the pathogenesis of diabetic nephropathy. We previously demonstrated that up-regulation of type II TGF-β receptor (TβRII) induced by high glucose might contribute to distal tubular hypertrophy [Yang, Guh, Yang, Lai, Tsai, Hung, Chang and Chuang (1998) J. Am. Soc. Nephrol. 9, 182–193]. We have elucidated the mechanism by using cultured Madin–Darby canine kidney cells. Enhancer assay and electrophoretic-mobility-shift assay were used to estimate the involvement of transcription factors. Western blotting and an in vitro kinase assay were used to evaluate the level and activity of protein kinase. We showed that glucose (100–900 mg/dl) induced an increase in mRNA level and promoter activity of TβRII (note: ‘mg/dl’ are the units commonly used in diabetes studies). The promoter region −209 to −177 appeared to contribute to positive transactivation of TβRII promoter by comparing five TβRII–promoter–CAT (chloramphenicol acetyl-transferase) plasmids. Moreover, the transcription factor AP-1 (activator protein 1) was significantly activated and specifically binds to TβRII promoter (−209 to −177). More importantly, we found that atypical PKC ι might be pivotal for high glucose-induced increase in both AP-1 binding and TβRII promoter activity. First, high glucose induced cytosolic translocation, activation and autophosphorylation of PKC ι. Secondly, antisense PKC ι expression plasmids attenuated high-glucose-induced increase in AP-1 binding and TβRII promoter activity; moreover, sense PKC ι expression plasmids enhanced these instead. Finally, we showed that antisense PKC ι expression plasmids might partly attenuate a high-glucose/TGF-β1-induced increase in fibronectin. We conclude that PKC ι might mediate high-glucose-induced increase in TβRII promoter activity. In addition, antisense PKC ι expression plasmid effectively suppressed up-regulation of TβRII and fibronectin in hyperglycaemic distal-tubule cells.
Functional characterization and regulation of the taurine transporter and cysteine dioxygenase in human hepatoblastoma HepG2 cells
Authors: SATSU Hideo , TERASAWA Eriko , HOSOKAWA Yu , SHIMIZU Makoto
Published: October 2003

We investigated the characterization and the regulation of TAUT (taurine transporter) and CDO (cysteine dioxygenase), one of the key enzymes of taurine biosynthesis, in human hepatoblastoma HepG2 cells. The activity of TAUT in the HepG2 cells was evaluated by means of a sodium- and chloride-dependent high-affinity transport system, the characteristics of which were similar to those of the β amino-acid-specific taurine transport system described previously for various tissues [Uchida, Kwon, Yamauchi, Preston, Marumo and Handler (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8230–8234; Ramamoorthy, Leibach, Mahesh, Han, Yang-Feng, Blakely and Ganapathy (1994) Biochem. J. 300, 893–900; and Satsu, Watanabe, Arai and Shimizu (1997) J. Biochem. (Tokyo) 121, 1082–1087]. By culturing in a hypertonic medium, the intracellular taurine content of HepG2 cells was markedly increased. Under hypertonic conditions, the activity of TAUT was up-regulated, and the results of the kinetic analysis suggested that this up-regulation was associated with an increase in the amount of TAUT. The expression level of TAUT mRNA was markedly higher than that of the control cells. The expression level of CDO mRNA was also up-regulated under the hypertonic conditions. Culturing the cells in a taurine-rich medium resulted in both the activity of TAUT and the expression level of TAUT mRNA being down-regulated in HepG2 cells. On the other hand, the expression level of CDO mRNA was not affected under a taurine-rich condition. The present results show that both TAUT and CDO were co-operatively regulated in response to hypertonicity, but did not co-operatively respond to the change in extracellular taurine concentration. Generally, the TAUT and taurine biosynthetic enzymes have independent regulatory systems, but under certain conditions, they could be regulated in harmony with each other.
Investigating interactions of the pentraxins serum amyloid P component and C-reactive protein by mass spectrometry
Authors: AQUILINA J. Andrew , ROBINSON Carol V.
Published: October 2003

The oligomeric state of human SAP (serum amyloid P component) in the absence and presence of known ligands has been investigated using nanoelectrospray ionization MS. At pH 8.0, in the absence of Ca2+, SAP has been shown to consist of pentameric and decameric forms. In the presence of physiological levels of Ca2+, SAP was observed to exist primarily as a pentamer, reflecting its in vivo state. dAMP was shown not only to promote decamerization, but also to lead to decamer stacking involving up to 30 monomers. A mechanism for this finding is proposed. CRP (C-reactive protein), a pentraxin closely related to SAP, exists as a pentamer in the presence or absence of Ca2+. Pentamers of CRP and SAP were shown to form mixed decamers in Ca2+-free buffer; however, in the presence of Ca2+, this interaction was not observed. Furthermore, no exchange of monomeric subunits was observed between the SAP and CRP oligomers, suggesting a remarkable stability of the individual pentameric complexes.
Cyclic ADP-ribose is a second messenger in the lipopolysaccharide-stimulated proliferation of human peripheral blood mononuclear cells
Authors: BRUZZONE Santina , DE FLORA Antonio , USAI Cesare , GRAEFF Richard , LEE Hon Cheung
Published: October 2003

Cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, was recently demonstrated to stimulate proliferation of various cell types. The role of cADPR in a specific process of monocyte- and plasma-mediated activation of T-lymphocytes by lipopolysaccharide (LPS) was addressed using human mononuclear cells from peripheral blood (PBMCs). Incubation of PBMCs with 0.1 μg/ml of LPS for 24 h provided a doubling in the intracellular levels of cADPR as compared with unstimulated PBMCs. The cADPR increase was abolished either by prior removal of monocytes or by pre-incubating a whole PBMC population with a monoclonal antibody against the monocyte marker CD14. The increased concentrations of intracellular cADPR elicited by LPS stimulation were paralleled by significant increases in NAD+ levels and in the activities of ectocellular and membrane-bound fractions of ADP-ribosyl cyclase/cADPR hydrolase activities. A cytosolic ADP-ribosyl cyclase was also detectable in PBMCs and its activity was comparably enhanced by LPS stimulation. This soluble cyclase is distinguished from the membrane-bound cyclase by both substrate and inhibitor sensitivities. LPS-stimulated PBMCs showed 2–3-fold increases of intracellular calcium ([Ca2+]i), and these changes were prevented completely by the cADPR antagonist 8-Br-cADPR and by ryanodine. Both compounds, and the cyclase inhibitor nicotinamide, significantly inhibited the T-lymphocyte proliferation induced by LPS in PBMCs. These results demonstrate that cADPR plays a role of second messenger in the adaptive immune recognition process of LPS-stimulated proliferation of PBMCs.
Molecular characterization of the constitutive expression of the plasma platelet-activating factor acetylhydrolase gene in macrophages
Authors: WU Xiaoqing , McINTYRE Thomas M. , ZIMMERMAN Guy A. , PRESCOTT Stephen M. , STAFFORINI Diana M.
Published: October 2003

Plasma platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase that inactivates platelet-activating factor (PAF) and PAF-like lipids to generate products with little or no biological activity. The levels of circulating PAF-AH correlate with several disease syndromes. We previously reported that mediators of inflammation regulate the expression of the human PAF-AH gene at the transcriptional level. In the present paper, we characterize the constitutive expression of plasma PAF-AH using the mouse gene as a model system, and we report comparative results obtained using human and mouse promoter constructs. We first cloned, sequenced and analysed the promoter region of the murine plasma PAF-AH (mPAF-AH) gene and found that this gene lacks a canonical TATA box. We demonstrated that the cis-elements required for basal transcription are localized within the −316 to −68 bp region. In vitro band-shift and supershift assays showed that Sp1 and Sp3 transcription factors from RAW264.7 and J774A.1 macrophage nuclear extracts bound strongly to a distal GC-rich site within −278/−243 [specificity protein (Sp-A)] and to a proximal TC-rich motif within −150/−114 (Sp-B). In addition, we observed weak binding to a GA-rich site within −110/−82 (Sp-C). The regions containing Sp-B and Sp-C are highly conserved between the human and mouse genes. Forced expression of Sp1 or Sp3 in Sp-lacking Drosophila SL2 cells induced markedly the activity of the exogenous mPAF-AH promoter in a dose-dependent manner, and this induction was dependent on the presence of intact Sp-A and Sp-B. Interestingly, we found that the Sp1- and Sp3-associated DNA-binding activities increased during the maturation of primary human monocytes into macrophages in cell culture. These results demonstrate that Sp1 and Sp3 are key factors that contribute to the basal, constitutive transcription of the plasma PAF-AH gene in macrophages.
Fibrillin-1 and −2 contain heparin-binding sites important for matrix deposition and that support cell attachment
Authors: RITTY Timothy M. , BROEKELMANN Thomas J. , WERNECK Claudio C. , MECHAM Robert P.
Published: October 2003

Fibrillin-1 and −2 are large modular extracellular matrix glycoproteins found in many vertebrate organ systems and are known to be key components of the elastic fibre. In the present study, we identify a new heparin-binding region in fibrillin-2 between exons 18 and 24. Additionally, we have narrowed the location of heparin-binding activity previously identified in fibrillin-1 to the last 17 residues of the mature proteolytically processed protein. This domain demonstrated higher activity as a multimer than as a monomer. The fibrillin-1 C-terminal site supported cell attachment in each of nine cell types tested. Attachment was shown to be mediated by cell-surface heparan sulphate proteoglycans. Fibrillin-1 has been shown previously to have heparin-binding activity that is important for matrix deposition of the molecule by fibroblasts. This function in deposition was confirmed in two additional fibrillin-producing cell types (osteosarcoma and epithelial cells) for the deposition of both fibrillin-1 and −2 into the extracellular matrix.
Beneficial effect of oleoylated lipids on paraoxonase 1: protection against oxidative inactivation and stabilization
Authors: NGUYEN S. Duy , SOK Dai-Eun
Published: October 2003

The effect of lipids on PON1 (paraoxonase 1), one of antioxidant proteins in high-density lipoprotein, was investigated in respect to inhibition, protection against oxidative inactivation, and stabilization. When the effect of lipids on the PON1 activity was examined, a remarkable inhibition was expressed by polyenoic fatty acids (C18:2–C20:5). Linoleic acid, the most potent (Ki, 3.8 μM), showed competitive inhibition. Next, various lipids were examined for prevention against the inactivation of PON1 by ascorbate/Cu2+, which caused a remarkable (≥90%) inactivation of PON1. Compared with saturated fatty acids (C6–C18), exhibiting a modest protection (9–40%), monoenoic acids (C16:1–C20:1) showed a greater maximal protective effect (Emax, 70–82%), with oleic acid being the most effective (EC50, 2.7 μM). In contrast, polyenoic acids showed no protection. Noteworthy, linoleic acid prohibited the protective action of oleic acid non-competitively. In the structure–activity relationship, a negatively charged group seems to be required for the protective action. Consistent with this, dioleoylphosphatidylglycerol, negatively charged, was more protective than dioleoylphosphatidylcholine. These data, together with requirement of Ca2+ (EC50, 0.6 μM) for the protective action, may support the existence of a specific site responsible for the protective action. A similar protective action of lipids was also observed in the inactivation of PON1 by ascorbate/Fe2+, peroxides or p-hydroxymercuribenzoate. Separately, PON1 was stabilized by oleic acid or oleoylated phospholipids, in combination with Ca2+, but not linoleic acid. These results suggest that in contrast to an adverse action of linoleic acid, monoenoic acids or their phospholipid derivatives play a beneficial role in protecting PON1 from oxidative inactivation as well as in stabilizing PON1.
Graminicide insensitivity correlates with herbicide-binding co-operativity on acetyl-CoA carboxylase isoforms
Authors: PRICE Lindsey J. , HERBERT Derek , MOSS Stephen R. , COLE David J. , HARWOOD John L.
Published: October 2003

The sensitivity of grass species to important classes of graminicide herbicides inhibiting ACCase (acetyl-CoA carboxylase) is associated with a specific inhibition of the multifunctional ACCase located in the plastids of grasses. In contrast, the multisubunit form of ACCase found in the chloroplasts of dicotyledonous plants is insensitive and the minor cytosolic multifunctional isoforms of the enzyme in both types of plants are also less sensitive to inhibition. We have isolated, separated and characterized the multifunctional ACCase isoforms found in exceptional examples of grasses that are either inherently insensitive to these graminicides, or from biotypes showing acquired resistance to their use. Major and minor multifunctional enzymes were isolated from cell suspension cultures of Festuca rubra and the ‘Notts A1’-resistant biotype of Alopecurus myosuroides, and their properties compared with those isolated from cells of wild-type sensitive A. myosuroides or from sensitive maize. Purifications of up to 300-fold were necessary to separate the two isoforms. The molecular masses (200–230 kDa) and Km values for all three substrates (ATP, bicarbonate and acetyl-CoA) were similar for the different ACCases, irrespective of their graminicide sensitivity. Moreover, we found no correlation between the ability of isoforms to carboxylate propionyl-CoA and their sensitivity to graminicides. However, insensitive purified forms of ACCase were characterized by herbicide-binding co-operativity, whereas, in contrast, sensitive forms of the enzymes were not. Our studies on isolated individual isoforms of ACCase from grasses support and extend previous indications that herbicide binding co-operativity is the only kinetic property that differentiates naturally or selected insensitive enzymes from the typical sensitive forms usually found in grasses.
Isolation and identification of l-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system
Authors: WAFA Latif A. , CHENG Helen , RAO Mira A. , NELSON Colleen C. , COX Michael , HIRST Martin , SADOWSKI Ivan , RENNIE Paul S.
Published: October 2003

The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate. Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins. However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity. To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD. DDC (l-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo. Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide. Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected. Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression.
Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of β2-chimaerin, a ‘non-protein kinase C’ phorbol ester receptor
Authors: CALOCA Maria Jose , WANG HongBin , KAZANIETZ Marcelo G.
Published: October 2003

The regulation and function of β2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to β2-chimaerin with high affinity and promote its subcellular distribution. β2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. β2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the β2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of β2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor β2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.
Differences in properties between human αA- and αB-crystallin proteins expressed in Escherichia coli cells in response to cold and extreme pH
Authors: TAKEUCHI Satoru , MANDAI Yumi , OTSU Akiko , SHIRAKAWA Taro , MASUDA Katsuyoshi , CHINAMI Masanobu
Published: October 2003

It has been reported that αA-crystallin has greater protective effects against apoptosis in lens epithelial cells than αB-crystallin [Andley, Song, Wawrousek, Fleming and Bassnett (2000) J. Biol. Chem. 275, 36823–36831]. Because the αA-crystallin proteins are specifically expressed in the vertebrate lens, we examine the non-specific properties of both αA- and αB-crystallins in an Escherichia coli system. E. coli cells were transformed with the inducible protein expression vector pET-11a, harbouring the gene for either human αA- or αB-crystallin, and two other control plasmids, pET-1la vector alone or pGEX-2T vector encoding GST (glutathione S-transferase). These cells were exposed to various stress conditions, such as cold-shock at 4 °C or extremely low or high pH environments (pH 4.7 or pH 8.0) for 6 h, and survival of the host cells and the solubility of the expressed target proteins in the cytosol were examined. Under these stress conditions, the cells expressing αB-crystallin protein demonstrated significantly improved survival when compared with the other cells, and the expressed protein in the cytosol was almost soluble, in contrast with the αA-crystallin protein. Differences in the amino acid sequence between the proteins in a phenylalanine-rich region next to the N-terminal consensus α-crystallin domain was considered to be responsible for chaperone activity and cell survival.
Processing, localization and binding activity of zonadhesin suggest a function in sperm adhesion to the zona pellucida during exocytosis of the acrosome
Authors: BI Ming , HICKOX John R. , WINFREY Virginia P. , OLSON Gary E. , HARDY Daniel M.
Published: October 2003

Zonadhesin is a sperm protein that binds in a species-specific manner to the extracellular matrix ZP (zona pellucida) of the mammalian oocyte. The pig zonadhesin precursor is a 267000-Da mosaic protein with a Type I membrane topology and a large extracellular region comprising meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and five tandem von Willebrand D (VWD) domains. Multiple mature forms of zonadhesin in the sperm head differ in their avidities for the ZP. To determine the potential functions of zonadhesin forms in gamete adhesion, we characterized the processing, activation and localization of protein in pig spermatozoa. The predominant polypeptides of processed zonadhesin were Mr 300000 (p300), 105000 (p105) and 45000 (p45). p45 and p105, comprised primarily the D1, D2–D3 domains respectively, and were N-glycosylated. p300 was heavily O-glycosylated, and spanned the meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and D0 domains. Hydrolysis of the precursor polypeptide occurred in the testis, and N-terminal sequencing of p45 and p105 identified Asp806-Pro and Asp1191-Pro in the D1 and D2 domains respectively as bonds cleaved in the protein's functional maturation. Testicular zonadhesin was extractable with non-ionic detergents, and localized to the developing outer acrosomal membrane of round and elongating spermatids. As spermatozoa transited the epididymis, most of the protein became incorporated into an extraction-resistant fraction, and the proportions of active and of multimeric zonadhesins in the cells increased. Zonadhesin localized to the perimeter of the acrosome in intact ejaculated spermatozoa and to the leading edge of acrosomal matrix overlying cells with disrupted acrosomal membranes. We conclude that the zonadhesin precursor is specifically proteolysed, glycosylated and assembled into particulate structures in the distal parts of the acrosome where it may mediate specific adhesion to the ZP during the initial stages of acrosomal exocytosis.
Lipid binding and membrane penetration of polymyxin B derivatives studied in a biomimetic vesicle system
Authors: KATZ Marina , TSUBERY Haim , KOLUSHEVA Sofiya , SHAMES Alex , FRIDKIN Mati , JELINEK Raz
Published: October 2003

Understanding membrane interactions and cell-wall permeation of Gram-negative bacteria is of great importance, owing to increasing bacterial resistance to existing drugs and therapeutic treatments. Here we use biomimetic lipid vesicles to analyse membrane association and penetration by synthetic derivatives of polymyxin B (PMB), a potent naturally occurring antibacterial cyclic peptide. The PMB analogues studied were PMB nonapeptide (PMBN), in which the hydrophobic alkyl residue was cleaved, PMBN diastereomer containing d-instead of l-amino acids within the cyclic ring (dPMBN) and PMBN where the hydrophobic alkyl chain was replaced with an Ala6 repeat (Ala6-PMBN). Peptide binding measurements, colorimetric transitions induced within the vesicles, fluorescence quenching experiments and ESR spectroscopy were applied to investigate the structural parameters underlying the different membrane-permeation profiles and biological activities of the analogues. The experiments point to the role of negatively charged lipids in membrane binding and confirm the prominence of lipopolisaccharide (LPS) in promoting membrane association and penetration by the peptides. Examination of the lipid interactions of the PMB derivatives shows that the cyclic moiety of PMB is not only implicated in lipid attachment and LPS binding, but also affects penetration into the inner bilayer core. The addition of the Ala6 peptide moiety, however, does not significantly promote peptide insertion into the hydrophobic lipid environment. The data also indicate that the extent of penetration into the lipid bilayer is not related to the overall affinity of the peptides to the membrane.
SCP1 encodes an actin-bundling protein in yeast
Authors: WINDER Steven J. , JESS Thomas , AYSCOUGH Kathryn R.
Published: October 2003

The association of F-actin (filamentous actin) with a large number of binding proteins is essential for cellular function. Actin-binding proteins control the dynamics of actin filaments, nucleate new filaments and facilitate formation of higher-order structures such as actin bundles. The yeast gene SCP1 encodes a small protein with significant homology to mammalian SM22/transgelin. We have investigated the role of Scp1p in budding yeast to probe the fundamental role of this family of proteins. Here, we demonstrate that Scp1p binds to F-actin and induces the formation of tight F-actin bundles in vitro. Deletion of SCP1 in yeast lacking the actin-bundling protein, fimbrin (Sac6p), exacerbates the disrupted actin phenotype and enhances latrunculin-A sensitivity. Furthermore, Scp1p co-localizes with actin in cortical patches and its localization is lost in the presence of latrunculin-A. Our data support a role for Scp1p in bundling actin filaments and, in concert with Sac6p, acting as a second actin-bundling activity crucial to the stability of the yeast actin cytoskeleton.
Direct interaction of β-dystroglycan with F-actin
Authors: CHEN Yun-Ju , SPENCE Heather J. , CAMERON Jacqueline M. , JESS Thomas , ILSLEY Jane L. , WINDER Steven J.
Published: October 2003

Dystroglycans are essential transmembrane adhesion receptors for laminin. α-Dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of β-dystroglycan. β-Dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin–glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of β-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells.
Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus
Authors: KANUGULA Sreenivas , PEGG Anthony E.
Published: October 2003

AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from two extremely thermophilic organisms, the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus were PCR-derived and cloned into an expression vector. The nucleotide sequence of the Aq. aeolicus AGT encodes a 201-amino-acid protein with a molecular mass of 23000 Da and Ar. fulgidus AGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. The Aq. aeolicus and Ar. fulgidus AGTs were expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairing O6-methylguanine and, at a lower rate, O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that from Aq. aeolicus, were readily inactivated by the low-molecular-mass inhibitor O6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy.
Zinc suppresses the iron-accumulation phenotype of Saccharomyces cerevisiae lacking the yeast frataxin homologue (Yfh1)
Authors: SANTOS Renata , DANCIS Andrew , EIDE David , CAMADRO Jean-Michel , LESUISSE Emmanuel
Published: October 2003

Analysis of Saccharomyces cerevisiae cell transcriptome revealed that iron deprivation/supplementation affects genes other than those of the iron regulon (controlled by Aft proteins). Several genes regulated by zinc (induced by zinc deprivation) were induced by iron. Cells lacking the yeast frataxin homologue Yfh1 accumulate large amounts of iron in their mitochondria. We have shown that the zinc metabolism of these cells is also impaired: zinc uptake and zinc accumulation were both much lower in Δyfh1 cells than in wild-type cells. Excess zinc in the growth medium also influenced the phenotypes of Δyfh1 cells. It prevented the accumulation of iron in the mitochondria of Δyfh1 cells and increased the growth rate of these cells and their resistance to oxidative stress. However, zinc did not restore the deficiency of Fe–S and haem proteins of Δyfh1 cells. Zinc inhibited mitochondrial respiration and protected Yah1p, the mitochondrial ferredoxin. These results suggest that zinc nutrition may be important in the aetiology of Friedreich's ataxia.
Bcl-2 activates a programme of premature senescence in human carcinoma cells
Authors: CRESCENZI Elvira , PALUMBO Giuseppe , BRADY Hugh J. M.
Published: October 2003

The apoptosis regulator Bcl-2 has been shown to modulate cell-cycle progression, favouring a quiescent state over a proliferative state, in both normal and tumour cells. We show here that constitutive expression of Bcl-2 in human carcinoma cells results in a cell-cycle arrest that within a few days can become irreversible. Arrested cells acquire a senescent-like phenotype, which consists of several characteristic morphological alterations and increased activity of senescence-associated β-galactosidase. The induction of the premature senescence programme is mediated by inhibition of Cdk2 kinase activity, and p27KIP1 is required to maintain the senescent phenotype. We propose that the ability to activate an endogenous premature senescence programme allows Bcl-2 to suppress tumour growth. These results suggest that the down-regulation of Bcl-2 expression, which has been observed during the development and progression of human carcinoma, is related to the ability of Bcl-2 to severely hamper the growth of carcinoma cells and to induce a permanent cell-cycle arrest, with the features of senescence.
Selective modulation of the SM22α promoter by the binding of BTEB3 (basal transcription element-binding protein 3) to TGGG repeats
Authors: MARTIN Karen M. , ELLIS Peter D. , METCALFE James C. , KEMP Paul R.
Published: October 2003

We have previously identified a C2H2 zinc-finger transcription factor [BTEB3 (basal transcription element-binding protein 3)/KLF13 (Krüppel-like factor 13)] that activates the minimal promoter for the smooth muscle-specific SM22α gene in other types of cell. We show that recombinant BTEB3 binds to three TGGG motifs in the minimal SM22α promoter. By mutation analysis, only one of these boxes is required for BTEB3-dependent promoter activation in P19 cells and BTEB3 activates or inhibits reporter gene expression depending on the TGGG box to which it binds. Transient transfection experiments show that BTEB3 also activates reporter gene expression from the SM22α promoter in VSMCs (vascular smooth muscle cells). Similar studies showed that BTEB3 did not activate expression from the promoter regions of the smooth muscle myosin heavy chain or smooth muscle α-actin promoters, which contain similar sequences, implying that promoter activation by BTEB3 is selective. The expression of BTEB3 is readily detectable in VSMCs in vitro and is modulated in response to injury in vivo.
Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition
Authors: KOMANDER David , KULAR Gursant S. , BAIN Jennifer , ELLIOTT Matthew , ALESSI Dario R. , van AALTEN Daan M. F.
Published: October 2003

PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors.
Pheochromocytoma associated with pregnancy
Authors: Kalra Jaswinder K. , Jain Vanita , Bagga Rashmi , Gopalan Sarala , Bhansali Anil K. , Behera Arunanshu , Batra Yatinder K.
Published: October 2003

Pheochromocytoma associated with pregnancy is rare with potentially lethal consequences. Antepartum diagnosis improves the maternal and perinatal outcome. The issue of mode of delivery is unresolved. Its definitive treatment is surgical resection preceded by medical management. Surgical resection may be done during caesarean section as is reported in the present case.
Ovarian endodermal sinus tumor in a 76‐year‐old woman
Authors: Filiz Gülaydan , Ozuysal Sema , Bilgin Tufan
Published: October 2003

A 76‐year‐old woman underwent surgery for pelvic mass, during which a 13 × 8‐cm right ovarian tumor was discovered. On histopathological examination, she was diagnosed with an endodermal sinus tumor with right tubal metastasis. The patient was treated with four cycles of Bleomycin, Etoposide and Cisplatin. She died of disseminated disease four years later.
Cotyledonary‐like placenta associated with severe intrauterine growth restriction
Authors: Kita Naoko , Ohira Satoshi , Ashida Takashi , Kanai Makoto , Mitsuhashi Yuko , Konishi Ikuo
Published: October 2003

There have been no previous reports of human placentas mimicking the cotyledonary placenta of ruminants. We report a case of cotyledonary‐like placenta associated with severe intrauterine growth restriction.

A woman pregnant for the first time was referred to our hospital at 27 weeks’ gestation because of severe intrauterine growth restriction. Characteristics of the placenta in the patient were examined by ultrasonographical and histopathological techniques. Ultrasonography revealed that a hypo‐echoic area intermingled with small segmented tissues in the placenta. The hypo‐echoic area changed in size and shape according to uterine contractions. At 31 weeks’ gestation, a caesarean section was performed because of non‐reassuring fetal status. A female baby weighing 814 g was delivered. The placenta, which weighed 260 g, contained several long stem villi running over a distance of 6 to 7 cm without branching into intermediate or terminal villi. Small villous tissues had developed only at the tips of these stem villi. The present case shows an extraordinarily abnormal development of the villous trees in the placenta, which may have caused fetal IUGR and non‐reassuring fetal status.
Complete hydatidiform mole in a triplet pregnancy coexisting two viable fetuses: Case report and review of the literature
Authors: Takagi Kimiyo , Unno Nobuya , Hyodo Hiro‐e M. , Hyodo Hironobu , Kashima Hiroyasu , Kubota Noriko , Ogiso Yoshihumi , Noike Masami , Itoh Kazuko , Shiobara Shigeki , Konishi Ikuo , Saito Shigeru
Published: October 2003

We report a rare case of a complete hydatidiform mole with two or more coexisting fetuses where both infants survived without complications. A male infant weighing 1258 g and a female infant weighing 880 g were delivered without complications and discharged 95 days after the birth. The analysis of DNA microsatellite polymorphisms indicated that the mole was of paternal origin and probably homozygous. The mother suffered from multiple pulmonary metastasis of the hydatidiform mole which was detected 3 days after the surgery and was successfully treated with methotrexate. A complete hydatidiform mole with two or more coexisting fetuses produces a dilemma between immediate termination and pregnancy continuation. Although the present case resulted in a favorable outcome, a review of the 14 reported cases suggests that the high fetal loss rate (90%) must be a consideration in the decision regarding management of such a pregnancy.
Cardiac failure caused by severe pre‐eclampsia with placental abruption, and its treatment with anti‐hypertensive drugs
Authors: Aoyama Kazufumi , Suzuki Yoshikatsu , Sato Takeshi , Yamamoto Tamao , Kojima Kazuhisa , Usami Takeshi , Ohte Nobuyuki , Suzumori Kaoru
Published: October 2003

Pre‐eclampsia is the abnormality of blood circulation in late pregnancy, often caused by renal failure, hemolysis, elevated liver enzyme, low platelet syndrome, and eclampsia. We present a case of severe pre‐eclampsia with placental abruption in a 24‐year‐old woman, pregnant for the first time. The patient was diagnosed with congestive heart failure, which came as a result of pre‐eclampsia. Anti‐hypertensive drugs were used for its treatment.
Preventable but not prevented: the reality of cervical cancer*
Authors: Saraiya Usha B.
Published: October 2003

Introduction: The incidence of invasive cervical cancer has decreased in the last 50 years in the developed countries substantially due to the use of routine pap smears. However, in the Asia–Oceanic region it continues to be high as screening programs are not established. Credit for starting cytology services in India goes to Professor P.N. Wahi of Agra. He became Founder President when about 34 cytologists got together in 1970 to form the Indian Academy of Cytologists. Since then cytology has spread through all parts of India. The Cytology Clinic in Cama & Albless Hospital was started in the same year. Since then over 100 000 women have been screened. Approximately 1200 cases of pre‐ and early cancers have been detected and treated. Since 1982 we are aware of the important role of human papillomavirus infection. We diagnose it by cytology and colposcopy and histology. Facilities for polymerase chain reaction, in‐situ hybridization and other virology studies are not available to us. CO2 laser treatment is found particularly useful in multicentric human papillomavirus disease.

Screening for the State of Maharashtra: Since 1984 we have planned for a screening program for our State. We have a population of 78.9 million. Approximately 15 million women in the age group of 35–64 years have to be screened. The health care infrastructure is good with 36 medical colleges and over 35 district hospitals. Screening is planned in phases. Trained personnel are the key to a successful program. In the final analysis, cervical cancer is not just a biomedical disease. It has socio‐cultural and economic implications.
Lipid peroxidation and vitamin E status in gestational diabetes mellitus
Authors: Santra Debjyoti , Sawhney Harjit , Aggarwal Neelam , Majumdar Siddarth , Vasishta Kala
Published: October 2003

Aim: To investigate any correlation between plasma levels of lipid peroxides, antioxidant nutrient (α‐tocopherol) and oxidized high‐density lipoprotein (HDL) in patients with gestational diabetes and those with a normal pregnancy and the incidence of pre‐eclampsia.

Methods: Sixty pregnant women attending an antenatal clinic were recruited for the study and were divided into two groups. Thirty women with gestational diabetes mellitus were recruited in the study group. The glucose‐tolerance‐test criteria, using 100 g of glucose taken orally, as laid down by the American College of Obstetricians and Gynecologists (1994) for diagnosis of gestational diabetes mellitus was used. Thirty gestation‐matched pregnant women with normal glucose tolerance test results were recruited as controls. A 10 mL venous blood sample was collected from each subject at the time of recruitment and thereafter at 4 week intervals until the time of delivery. Samples were analyzed for malondialdehyde thiobarbituric acid reactive, oxidized HDL‐cholesterol and α−tocopherol. The incidence of pre‐eclampsia and its correlation with antioxidant and lipid peroxide levels were compared in both the groups.

Results: Ten subjects out of 30 in the study group and three subjects out of 30 in the control group developed pre‐eclampsia. The incidence of preterm labor in both the groups was same (16.66%). The mean lipid peroxide level was lower in the study group at recruitment and later the levels kept falling, whereas levels of α−tocopherol and oxidized‐HDL were higher in the study group and kept on rising at follow up.

Conclusion: Gestational diabetes is not associated with increased levels of lipid peroxides and decreased levels of α‐tocopherol.