bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of nisin production by Lactococcus lactis in periodically re‐alkalized cultures
Authors: Guerra Nelson Pérez , Castro Lorenzo Pastrana
Published: October 2003

Synthesis of nisin as well as biomass production by Lactococcus lactis subsp. lactis CECT (Colección Española de Cultivos Tipo) 539 on both hydrolysed mussel‐processing waste and whey medium were followed in three fixed volume fed‐batch fermentations, with re‐alkalization cycles. The two cultures on mussel‐processing waste (MPW) were fed with a 240 g/l concentrated glucose and with a concentrated MPW (about 100 g of glucose/l). The culture on whey was fed with a mixture of concentrated whey (48 g of total sugars/l) and a 400 g/l concentrated lactose. The three cultures were mainly characterized with higher nisin titres [49.7, 109.6 and 124.7 bacteriocin activity units (AU)/ml respectively] compared with the batch process on de Man, Rogosa and Sharpe [(1960) J. Appl. Bacteriol. 23, 130–135] medium (49.6 AU/ml), MPW (9.5 AU/ml) and whey (22.5 AU/ml) [1 AU/ml is the amount of antibacterial compound needed to obtain 50% growth inhibition (LD50) compared with control tubes]. In the three fed‐batch cultures a shift from homolactic to mixed‐acid fermentation was observed, and other products (acetic acid, butane‐2,3‐diol or ethanol) in addition to lactic acid were detectable in the medium. However, their contributions to the total antibacterial activity of the post‐incubates (the cell‐free culture supernatant obtained at the end of the fermentation process) of L. lactis CECT 539 against Carnobacterium piscicola CECT 4020 were very low.
CO2 hydration by immobilized carbonic anhydrase
Authors: Bhattacharya Sumana , Schiavone Marc , Chakrabarti Subhra , Bhattacharya Sanjoy K.
Published: October 2003

The enzyme carbonic anhydrase (isoform II) from bovine and human erythrocytes was immobilized using different covalent coupling methods on inert matrices. Immobilized carbonic anhydrase may enable concentration of CO2 for Rubisco (ribulose‐1,5‐bisphosphate carboxylase/oxygenase)‐catalysed fixation in bioreactors. In the present study the activity of carbonic anhydrase with respect to hydration of CO2 using soluble and immobilized enzymes was determined. The stability of the immobilization matrix, the properties of the immobilized enzymes subjected to a variation in operation variables and the activity profile with respect to storage are reported. Immobilization imparted greater thermal and storage stability and enhanced reusability.
Lipase‐catalysed enantioselective ammonolysis of phenylglycine methyl ester in organic solvent
Authors: Du Wei , Zong Minhua , Guo Yong , Liu Dehua
Published: October 2003

Ammonium, provided by ammonium carbamate as a novel acyl acceptor, was adopted for enzymic enantio‐selective ammonolysis of racemic phenylglycine methyl ester in this paper and it has been found that the reaction conditions have profound effects on enzymic activity and enantioselectivity: the optimal concentration of ammonium carbamate was 80 mM; t‐butanol was the most suitable reaction medium and the optimum initial water activity was found to be 0.75; relatively high reaction rate and enantioselectivity could be attained within the temperature range of 30–40 °C. Compared with the corresponding hydrolysis and alcoholysis, enzymic ammonolysis expressed rather high enzymic activity and enantioselectivity and 95% of enzymic activity remained after 10 batches of ammonolysis, which demonstrates that lipase‐catalysed ammonolysis is a promising method for the preparation of optically pure (R)‐phenylglycine and its derivatives.
Caeruloplasmin sterilized by γ‐irradiation in the presence of l‐tyrosine maintains structural and catalytic characteristics
Authors: Assemand Emma , Lacroix Monique , Mateescu Mircea‐Alexandru
Published: October 2003

It was previously shown that protein aggregation induced by γ‐irradiation can be prevented by l‐tyrosine, even when irradiation was done in solution at doses as high as 10 kGy. It is now reported that caeruloplasmin irradiated at low to moderate doses appears more resistant to trypsin‐mediated proteolysis than native caeruloplasmin. Ceruloplasmin presents an irradiation‐dose‐dependent decrease of oxidase activity and a slight increase of ferroxidase activity at low irradiation doses (2 kGy), followed by a decrease at high doses (4–8 kGy). In all cases, the catalytic activities are higher when caeruloplasmin is irradiated under tyrosine protection. Irradiated caeruloplasmin in the presence of tyrosine preserves its oxidase and ferroxidase activities. Caeruloplasmin irradiated in solution at a dose of 2–3 kGy and higher, in the presence and absence of l‐tyrosine, remained sterile during at least 8 weeks of storage at 4 °C, as evaluated by the plate‐count‐agar method for assessing total viable lactic acid bacteria content after incubation for 48 h at 30 °C.
Cytochrome c is reduced mainly by plastoquinol and not by superoxide in thylakoid membranes at low and medium light intensities: its specific interaction with thylakoid membrane lipids
Authors: KRUK Jerzy , JEMIOŁA-RZEMIŃSKA Małgorzata , STRZAŁKA Kazimierz
Published: October 2003

We have found that, at low light intensity (5–10 μmol photons·m−2·s−1), photoreduction of cyt (cytochrome) c by isolated thylakoids was not inhibited by dinitrophenylether of iodonitrothymol, an inhibitor of the cyt b6–f complex, and the inhibition was only partial at medium light intensity (50–200 μmol photons·m−2·s−1). The photoreduction was not significantly influenced by superoxide dismutase. The conclusion that cyt c could be reduced directly by the plastoquinone pool was confirmed by the observation that plastoquinol-9 reduced cyt c efficiently when it was incorporated into liposome membranes prepared from thylakoid membrane lipids. It was shown that the cyt is specifically bound to thylakoid lipid liposomes owing to the presence of negatively charged lipids, phosphatidylglycerol and sulphoquinovosyldiacylglycerol, and the reduction was stimulated by the presence of monogalactosyldiacylglycerol, an inverted micelles-forming lipid, in the membranes, where the cyt c reduction by plastoquinol probably takes place. The results obtained are also discussed in terms of reliability of the method of cyt c photoreduction for determining superoxide production by illuminated thylakoids.
Roles of 5′-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis
Authors: RUTTER Guy A. , da SILVA XAVIER Gabriela , LECLERC Isabelle
Published: October 2003

AMPK (5′-AMP-activated protein kinase) is emerging as a metabolic master switch, by which cells in both mammals and lower organisms sense and decode changes in energy status. Changes in AMPK activity have been shown to regulate glucose transport in muscle and glucose production by the liver. Moreover, AMPK appears to be a key regulator of at least one transcription factor linked to a monogenic form of diabetes mellitus. As a result, considerable efforts are now under way to explore the usefulness of AMPK as a therapeutic target for other forms of this disease. Here we review this topic, and discuss new findings which suggest that AMPK may play roles in regulating insulin release and the survival of pancreatic islet β-cells, and nutrient sensing by the brain.
Glucocorticoids suppress β-cell development and induce hepatic metaplasia in embryonic pancreas
Authors: SHEN Chia-Ning , SECKL Jonathan R. , SLACK Jonathan M. W. , TOSH David
Published: October 2003

Elevated glucocorticoids are associated with low birth weight and fetal ‘programming’ of hypertension and glucose intolerance. In the present paper, we show that treatment of fetal rats with dexamethasone during the last week of gestation reduces the insulin content of their pancreatic β-cells. We reproduce this effect of dexamethasone in vitro using organ cultures of mouse embryonic pancreas, and show that it is associated with an elevation of expression of the transcription factor C/EBPβ (CCAAT/enhancer-binding protein β) and a reduction of the transcription factor Pdx-1 (pancreatic duodenal homeobox-1). Dexamethasone also induces the appearance of hepatocyte-like cells in organ cultures of pancreas, based on the expression of liver markers, albumin, α1-antitrypsin and transthyretin. Evidence that C/EBPβ is responsible for compromising the differentiation and later function of β-cells is obtained from its effects on the β-cell-like cell line RIN-5F. Transfection with a constitutive form of C/EBPβ suppresses insulin formation, whereas introduction of a dominant-negative inhibitor of C/EBPβ has no effect. We conclude that dexamethasone inhibits insulin expression in pancreatic β-cells via a mechanism involving down-regulation of Pdx-1 and induction of C/EBPβ. This mechanism may operate in combination with other changes during fetal programming, leading to type 2 diabetes in later life.
Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions
Authors: FUERTES Graciela , DE LLANO José Javier MARTÍN , VILLARROYA Adoración , RIVETT A. Jennifer , KNECHT Erwin
Published: October 2003

The contribution of the main proteolytic pathways to the degradation of long-lived proteins in human fibroblasts grown under different conditions was investigated. The effects of various commonly used pharmacological inhibitors of protein degradation were first analysed in detail. By choosing specific inhibitors of lysosomes and proteasomes, it was observed that together both pathways accounted for 80% or more of the degradation of cell proteins. With lysosomal inhibitors, it was found that serum withdrawal or amino-acid deprivation strongly stimulated macroautophagy but not other lysosomal pathways, whereas confluent conditions had no effect on macroautophagy and slightly activated other lysosomal pathways. Prolonged (24 h) serum starvation of confluent cultures strongly decreased the macroautophagic pathway, whereas the activity of other lysosomal pathways increased. These changes correlated with electron microscopic observations and morphometric measurements of lysosomes. With proteasomal inhibitors, it was found that, in exponentially growing cells in the absence of serum, activity of the ubiquitin–proteasome pathway increases, whereas under confluent conditions the contribution (in percentage) of proteasomes to degradation decreases, especially in cells deprived of amino acids. Interestingly, in confluent cells, the levels of two components of the 19 S regulatory complex and those of an interchangeable β-subunit decreased. This was associated with a marked increase in the levels of components of PA28-immunoproteasomes. Thus confluent conditions affect proteasomes in a way that resembles treatment with interferon-γ. Altogether, these results show that the activity of the various proteolytic pathways depends on the growth conditions of cells and will be useful for investigation of the specific signals that control their activity.
Adaptor signalling proteins Grb2 and Grb7 are recruited by human G6f, a novel member of the immunoglobulin superfamily encoded in the MHC
Authors: de VET Edwin C. J. M. , AGUADO Begoña , CAMPBELL R. Duncan
Published: October 2003

The human G6f protein, which is encoded by a gene in the MHC, is a putative cell-surface receptor belonging to the immunoglobulin superfamily. The intracellular tail of G6f is 40 amino acids in length and contains one tyrosine residue (Y281), which is phosphorylated after treatment of cells with pervanadate. This tyrosine residue is found in a consensus-binding motif (YXN) for the Src homology 2 domains of Grb2 and Grb7 (where Grb stands for growth-factor-receptor-bound protein). Glutathione S-transferase pull-down assays showed that the interaction of G6f with both Grb2 and Grb7 is mediated through the Src homology 2 domains of these two proteins and is dependent on the phosphorylation of G6f. Immunoprecipitation experiments showed the interaction of full-length phosphorylated G6f with both full-length Grb2 and Grb7. Antibody cross-linking of G6f expressed in K562 cells resulted in a transient phosphorylation of p42/44 MAP kinase (also known as extracellular-signal-regulated protein kinase-1/2; MAP stands for mitogen-activated protein) which could be prevented by MAP kinase kinase (MEK) inhibitors. These results suggest a coupling of G6f with downstream signal transduction pathways involving Grb2 and Grb7, including the Ras–MAP kinase pathway.
Thrombin inhibits Bim (Bcl-2-interacting mediator of cell death) expression and prevents serum-withdrawal-induced apoptosis via protease-activated receptor 1
Authors: CHALMERS Claire J. , BALMANNO Kathryn , HADFIELD Kathryn , LEY Rebecca , COOK Simon J.
Published: October 2003

To investigate the role of thrombin in regulating apoptosis, we have used CCl39 cells, a fibroblast cell line in which thrombin-induced cell proliferation has been extensively studied. Withdrawal of serum from CCl39 cells resulted in a rapid apoptotic response that was completely prevented by the inclusion of thrombin. The protective effect of thrombin was reversed by pertussis toxin, suggesting that cell-survival signalling pathways are activated via a Gi or Go heterotrimeric GTPase. Serum-withdrawal-induced death required de novo gene expression and was preceded by the rapid de novo expression of the pro-apoptotic ‘BH3-only’ protein Bim (Bcl-2-interacting mediator of cell death). Thrombin strongly inhibited the up-regulation of both Bim protein and Bim mRNA. The ability of thrombin to repress Bim expression, and to protect cells from apoptosis, was reversed by U0126, a MEK1/2 [MAPK (mitogen-activated protein kinase) or ERK (extracellular-signal-regulated kinase) 1/2] inhibitor, or LY294002, a phosphoinositide 3′-kinase (PI3K) inhibitor, suggesting that both the Raf→MEK→ERK1/2 and PI3K pathways co-operate to repress Bim and promote cell survival. A PAR1p (protease-activated receptor 1 agonist peptide) was also able to protect cells from serum-withdrawal-induced apoptosis, suggesting that thrombin acts via PAR1 to prevent apoptosis.
Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide
Authors: OKADA Miki , TAKEZAWA Daisuke , TACHIBANAKI Shuji , KAWAMURA Satoru , TOKUMITSU Hiroshi , KOBAYASHI Ryoji
Published: October 2003

The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca2+-sensitive regulators, each with three Ca2+-binding EF-hand motifs. In plants, lily CCaMK [chimaeric Ca2+/CaM (calmodulin)-dependent protein kinase] and its PpCaMK (Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists, we screened a total of 43 compounds using Ca2+-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S100 proteins, in a Ca2+-dependent manner. Furthermore, the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC50 values of 400 μM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca2+-dependent manner and antagonized the regulatory function of the domain with IC50 values of 55 and 4 μM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked KATP channels with similar potency, glibenclamide had no antagonizing effect on the Ca2+-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca2+-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists.
Characterization of a potent human interleukin-11 agonist
Authors: HARMEGNIES Dimitri , WANG Xiao-Ming , VANDENBUSSCHE Paul , LEON Arnaud , VUSIO Patricia , GRÖTZINGER Joachim , JACQUES Yannick , GOORMAGHTIGH Erik , DEVREESE Bart , CONTENT Jean
Published: October 2003

Human interleukin-11 (hIL-11) is a multi-potential cytokine that is involved in numerous biological activities, such as haematopoiesis, osteoclastogenesis, neurogenesis and female fertility, and also displays anti-inflammatory properties. IL-11 is used clinically to treat chemotherapy-induced thrombocytopenia. Because of its broad spectrum of action, improved IL-11 agonists, as well as IL-11 antagonists, could be of interest for numerous clinical applications. IL-11 signalling is dependent on the formation of a tripartite ligand–receptor complex consisting of IL-11, the IL-11R (IL-11 receptor) α subunit (responsible for the specificity of the interaction) and gp130 (glycoprotein 130) receptor β subunit (responsible for signal transduction). The interaction between IL-11 and IL-11Rα subunit occurs at its recently assigned site I. We have designed an IL-11 mutein whose hydrophobicity at site I has been increased. The mutein has been characterized in terms of structure, affinity, specificity and bioactivity. Electrophoretic analysis, gel filtration, IR spectroscopy and CD indicate that this new protein is more compact than wild-type IL-11. It binds to IL-11Rα with a three-fold-enhanced affinity, and retains the ability to recruit gp130 through site II. However, analysis of its biological activity revealed a complex pattern: although this mutein is 60–400-fold more active than wild-type IL-11 on the proliferation of 7TD1 murine hybridoma cell, it is less active than IL-11 on the proliferation of B9 cells, another murine hybridoma cell line. The results are interpreted on the basis of an IL-11 conformational change induced by the mutations, and the preferential use by the mutein of another unknown transducing receptor chain.
Structure–function analysis of LIV-1, the breast cancer-associated protein that belongs to a new subfamily of zinc transporters
Authors: TAYLOR Kathryn M. , MORGAN Helen E. , JOHNSON Andrea , HADLEY Lisa J. , NICHOLSON Robert I.
Published: October 2003

The LIV-1 gene has been previously associated with oestrogen-positive breast cancer and its metastatic spread to the regional lymph nodes. We have investigated the protein product of this gene as a marker for disease progression of breast cancer. The protein sequence contains a potential metalloprotease motif (HEXPHEXGD), which fits the consensus sequence for the catalytic zinc-binding site motif of the zincin metalloproteases. This motif has identified a new subfamily of ZIP (Zrt-, Irt-like proteins) zinc transporters, which we have termed LZT (LIV-1 subfamily of ZIP zinc transporters). Expression of recombinant LIV-1 in Chinese-hamster ovary cells confirmed the prediction that LZT proteins can act as zinc-influx transporters. Zinc is essential for growth and zinc transporters have an important role in maintaining intracellular zinc homoeostasis, aberrations of which could lead to diseases such as cancer. This is the first report of the expression of a recombinant human LZT protein in mammalian cells. Recombinant LIV-1 locates to the plasma membrane, concentrated in lamellipodiae, similar to membrane-type metalloproteases. Examination of LIV-1 tissue expression located it mainly to hormonally controlled tissues with widespread expression in the brain. Interestingly, the LIV-1 sequence contains a strong PEST site and other potential degradation motifs, which, combined with our evidence that recombinant LIV-1 associates with ubiquitin, may explain the low-level expression of LIV-1. Combining the crucial role that zinc plays in cell growth and the proven role of metalloproteases in metastasis presents an exciting indication of how LIV-1 plays a role in breast cancer progression.
N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A
Authors: HENRIKSSON Hongbin , DENMAN Stuart E. , CAMPUZANO Iain D. G. , ADEMARK Pia , MASTER Emma R. , TEERI Tuula T. , BRUMER Harry
Published: October 2003

The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower (Brassica oleracea var. botrytis) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential N-linked glycosylation site, which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform, which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc2Man6. LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHDEIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40% decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated.
Regulation of cell-surface major histocompatibility complex class I expression by the endopeptidase EC3.4.24.15 (thimet oligopeptidase)
Authors: KIM Sandra I. , PABON Amanda , SWANSON Todd A. , GLUCKSMAN Marc J.
Published: October 2003

Endopeptidase EP24.15 (EC 3.4.24.15; thimet oligopeptidase), traditionally classified as a neuropeptide-processing enzyme, degrades well-known MHC I (major histocompatibility complex class I) peptides in cell extracts. In the present study, we determine the contribution of EP24.15 in vivo to the surface expression of MHC I on intact cells. CTLs (cytotoxic T-lymphocytes) recognize a vast array of peptides presented in the context of MHC I cell-surface molecules. Stable retroviral overexpression of EP24.15 induces a dramatic, long-term inhibition of surface MHC I. In contrast, overexpression of a mutant EP24.15, which is expressed, but is enzymically inactive, does not affect the surface MHC I expression level. We observed no difference in the effect of EP24.15 on the expression of different classes of MHC I. IFN-γ (interferon-γ) treatment re-established MHC I expression on these EP24.15-overexpressing cells, and also induced EP24.15 cytosolic protein expression and enzyme activity. To our knowledge, this is the first demonstration of cytokine-induced EP24.15 expression and activity. Conversely, stable retroviral silencing of endogenous EP24.15 by RNA interference induced a striking, long-term increase in surface MHC I. Subcellular fractionation and enzyme-activity experiments localized the vast majority of EP24.15 protein expression and function to the cytosol. Therefore we introduce a novel function of the cytosolic form of EP24.15. EP24.15 activity in the extracellular space is significant for neuropeptide processing, and in the present paper, we demonstrate that EP24.15 activity in the cytosol may be significant for regulation of MHC I cell-surface expression.
The mouse matrix metalloproteinase, epilysin (MMP-28), is alternatively spliced and processed by a furin-like proprotein convertase
Authors: ILLMAN Sara A. , KESKI-OJA Jorma , PARKS William C. , LOHI Jouko
Published: October 2003

Epilysin (MMP-28) is a recently identified member of the matrix metalloproteinase (MMP) family. To explore the expression of epilysin in vivo and to gain insight into its biological functions, we have cloned the mouse epilysin cDNA and determined its expression. The amino acid sequence of the mouse protein is 85% identical with the human sequence and contains conserved features such as an RKKR furin-activation sequence following the prodomain. Unexpectedly, we found two alternatively spliced forms of the epilysin mRNA lacking 30 and 72 nt at the beginning of the seventh exon coding for part of the haemopexin domain. Expression of recombinant epilysin in HT-1080 fibrosarcoma cells indicated that epilysin was secreted as a major 48 kDa form and a minor 58 kDa form. Expression of the 58 kDa form was increased by a synthetic furin inhibitor at the expense of the 48 kDa form, suggesting that furin cleaves and activates epilysin. Epilysin mRNA was detected in a number of mouse tissues, with the highest expression in the lung, placenta, heart and uterus, and lower levels in the testis and gastrointestinal tract. The wide expression of epilysin in intact, healthy tissues suggests that this MMP functions in physiological tissue homoeostasis and turnover.
Identification of SNARE complex modulators that inhibit exocytosis from an α-helix-constrained combinatorial library
Authors: BLANES-MIRA Clara , PASTOR Maria T. , VALERA Elvira , FERNÁNDEZ-BALLESTER Gregorio , MERINO Jaime M. , GUTIERREZ Luis M. , PEREZ-PAYÁ Enrique , FERRER-MONTIEL Antonio
Published: October 2003

Synthetic peptides patterned after the proteins involved in vesicle fusion [the so-called SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins] are potent inhibitors of SNARE complex assembly and neuronal exocytosis. It is noteworthy that the identification of peptide sequences not related to the SNARE proteins has not been accomplished yet; this is due, in part, to the structural constraints and the specificity of the protein interactions that govern the formation of the SNARE complex. Here we have addressed this question and used a combinatorial approach to identify peptides that modulate the assembly of the SNARE core complex and inhibit neuronal exocytosis. An α-helix-constrained, mixture-based, 17-mer combinatorial peptide library composed of 137180 sequences was synthesized in a positional scanning format. Peptide mixtures were assayed for their ability to prevent the formation of the in vitro-reconstituted SDS-resistant SNARE core complex. Library deconvolution identified eight peptides that inhibited the assembly of the SNARE core complex. Notably, the most potent 17-mer peptide (acetyl-SAAEAFAKLYAEAFAKG-NH2) abolished both Ca2+-evoked catecholamine secretion from detergent-permeabilized chromaffin cells and l-glutamate release from intact hippocampal primary cultures. Collectively, these findings indicate that amino acid sequences that prevent SNARE complex formation are not restricted to those that mimic domains of SNARE proteins, thus expanding the diversity of molecules that target neuronal exocytosis. Because of the implication of neurosecretion in the aetiology of several human neurological disorders, these newly identified peptides may be considered hits for the development of novel anti-spasmodic drugs.
Identification of new leishmanicidal peptide lead structures by automated real-time monitoring of changes in intracellular ATP
Authors: LUQUE-ORTEGA J. Román , SAUGAR José M. , CHIVA Cristina , ANDREU David , RIVAS Luis
Published: October 2003

Leishmanicidal drugs interacting stoichiometrically with parasite plasma membrane lipids, thus promoting permeability, have raised significant expectations for Leishmania chemotherapy due to their nil or very low induction of resistance. Inherent in this process is a decrease in intracellular ATP, either wasted by ionic pumps to restore membrane potential or directly leaked through larger membrane lesions caused by the drug. We have adapted a luminescence method for fast automated real-time monitoring of this process, using Leishmania donovani promastigotes transfected with a cytoplasmic luciferase form, previously tested for anti-mitochondrial drugs. The system was first assayed against a set of well-known membrane-active drugs [amphotericin B, nystatin, cecropin A–melittin peptide CA(1-8)M(1-18)], plus two ionophoric polyethers (narasin and salinomycin) not previously tested on Leishmania, then used to screen seven new cecropin A–melittin hybrid peptides. All membrane-active compounds showed a good correlation between inhibition of luminescence and leishmanicidal activity. Induction of membrane permeability was demonstrated by dissipation of membrane potential, SYTOXTM Green influx and membrane damage assessed by electron microscopy, except for the polyethers, where ATP decrease was due to inhibition of its mitochondrial synthesis. Five of the test peptides showed an ED50 around 1 μM on promastigotes. These peptides, with equal or better activity than 26-residue-long CA(1-8)M(1-18), are the shortest leishmanicidal peptides described so far, and validate our luminescence assay as a fast and cheap screening tool for membrane-active compounds.
The POU homeodomain protein OCT3 as a potential transcriptional activator for fibroblast growth factor-4 (FGF-4) in human breast cancer cells
Authors: WANG Peixiang , BRANCH Donald R. , BALI Meenakshi , SCHULTZ Gilbert A. , GOSS Paul E. , JIN Tianru
Published: October 2003

The POU (representing a homeodomain protein family of which the founder members are Pit-1, Oct-1/2 and Unc-86) homeodomain protein OCT3/Oct-3 (where OCT stands for octamer-binding protein) is an embryonic transcription factor expressed in oocytes, embryonic stem and embryonic carcinoma cells. We have demonstrated previously that human breast cancer cells regain the ability to express OCT3 mRNA [Jin, Branch, Zhang, Qi, Youngson and Goss (1999) Int. J. Cancer 81, 104–112]. Antibodies against human OCT3 were not available when this study was conducted. By using a human OCT3–glutathione S-transferase fusion protein to affinity purify a polyclonal antibody against the mouse Oct-3, we obtained an antibody that enabled us to detect OCT3 in human breast cancer cells by Western-blot analysis. Thus we have now confirmed that OCT3 is expressed in human breast cancer cells but not in normal human breasts and in three other organs. When breast cancer cell lines were treated with all-trans-retinoic acid, OCT3 expression was repressed, associated with decreased cell proliferation. Although another POU protein Brn-3 has been shown to be a repressor for BRCA1 (breast-cancer susceptibility gene 1), OCT3 does not repress human or mouse BRCA1/Brca-1 promoters. However, OCT3 is capable of activating a fusion promoter containing the fibroblast growth factor-4 (FGF-4) enhancer element. In addition, we documented for the first time that human breast cancer cells express FGF-4 protein, and its expression could be inhibited by all-trans-retinoic acid. Furthermore, overexpressing OCT3 stimulated endogenous FGF-4 expression in MCF7 breast cancer cell line. These observations indicate that OCT3 protein is selectively expressed in human breast cancer cells, and its expression may be implicated in mammary gland tumorigenesis via up-regulating FGF-4 expression.
Variations in aggrecan structure modulate its susceptibility to aggrecanases
Authors: ROUGHLEY Peter J. , BARNETT James , ZUO Fengrong , MORT John S.
Published: October 2003

Proteoglycan aggregates and purified aggrecan from adult and fetal bovine cartilage and adult and neonatal human cartilage were subjected to in vitro degradation by recombinant aggrecanase-1 and aggrecanase-2. The ability of the aggrecanases to cleave within the aggrecan IGD (interglobular domain) and CS2 domain (chondroitin sulphate-rich domain 2) was monitored by SDS/PAGE and immunoblotting. Aggrecanase-2 showed a similar ability to cleave within the IGD of adult and immature aggrecan, whereas aggrecanase-1 was less efficient in cleavage in the IGD of immature aggrecan, for both the bovine and the human substrates. Both aggrecanases showed a similar ability to cleave within the CS2 domain of bovine aggrecan irrespective of age, but showed a much lower ability to cleave within the CS2 domain of human aggrecan. Equivalent results were obtained whether aggrecan was present in isolation or as part of proteoglycan aggregates. When proteoglycan aggregates were used, neither aggrecanase was able to cleave link protein. Thus, for aggrecan cleavage by aggrecanases, variations in cleavage efficiency exist with respect to the species and age of the animal from which the aggrecan is derived and the type of aggrecanase being used.
Fructose modulates GLUT5 mRNA stability in differentiated Caco-2 cells: role of cAMP-signalling pathway and PABP (polyadenylated-binding protein)-interacting protein (Paip) 2
Authors: GOUYON Florence , ONESTO Cercina , DALET Veronique , PAGES Gilles , LETURQUE Armelle , BROT-LAROCHE Edith
Published: October 2003

In intestinal cells, levels of the fructose transporter GLUT5 are increased by glucose and to a greater extent by fructose. We investigated the mechanism by which fructose increases GLUT5 expression. In Caco-2 cells, fructose and glucose increased activity of the −2500/+41 GLUT5 promoter to the same extent. cAMP also activated the GLUT5 promoter. However, if a protein kinase A inhibitor was used to block cAMP signalling, extensive GLUT5 mRNA degradation was observed, with no change in basal transcription levels demonstrating the involvement of cAMP in GLUT5 mRNA stability. Indeed, the half-life of GLUT5 mRNA was correlated (R2=0.9913) with cellular cAMP levels. Fructose increased cAMP concentration more than glucose, accounting for the stronger effect of fructose when compared with that of glucose on GLUT5 production. We identified several complexes between GLUT5 3′-UTR RNA (where UTR stands for untranslated region) and cytosolic proteins that might participate in mRNA processing. Strong binding of a 140 kDa complex I was observed in sugar-deprived cells, with levels of binding lower in the presence of fructose and glucose by factors of 12 and 6 respectively. This may account for differences in the effects of fructose and glucose. In contrast, the amounts of two complexes of 96 and 48 kDa increased equally after stimulation with either glucose or fructose. Finally, PABP (polyadenylated-binding protein)-interacting protein 2, a destabilizing partner of PABP, was identified as a component of GLUT5 3′-UTR RNA–protein complexes. We conclude that the post-transcriptional regulation of GLUT5 by fructose involves increases in mRNA stability mediated by the cAMP pathway and Paip2 (PABP-interacting protein 2) binding.
Characterization of non-covalent oligomers of proteins treated with hypochlorous acid
Authors: CHAPMAN Anna L. P. , WINTERBOURN Christine C. , BRENNAN Stephen O. , JORDAN T. William , KETTLE Anthony J.
Published: October 2003

Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1–20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.
Binding of endostatin to endothelial heparan sulphate shows a differential requirement for specific sulphates
Authors: BLACKHALL Fiona H. , MERRY Catherine L. R. , LYON Malcolm , JAYSON Gordon C. , FOLKMAN Judah , JAVAHERIAN Kashi , GALLAGHER John T.
Published: October 2003

Endostatin is a naturally occurring proteolytic fragment of the C-terminal domain of collagen XVIII. It inhibits angiogenesis by a mechanism that appears to involve binding to HS (heparan sulphate). We have examined the molecular interaction between endostatin and HS from micro- and macrovessel endothelial cells. Two discrete panels of oligosaccharides were prepared from metabolically radiolabelled HS, using digestion with either heparinase I or III, and then examined for their endostatin affinity using a sensitive filter-binding assay. Two types of endostatin-binding regions were identified: one comprising sulphated domains of five or more disaccharides in length, enriched in 6-O-sulphate groups, and the other contained long heparinase I-resistant fragments. In the latter case, evidence from the present study suggests that the binding region encompasses a sulphated domain fragment and a transition zone of intermediate sulphation. The contribution to binding of specific O-sulphate groups was determined using selectively desulphated HS species, namely HS from Hs2st−/− mutant cells, and by comparing the compositions of endostatin-binding and non-binding oligosaccharides. The results indicate that 6-O-sulphates play a dominant role in site selectivity and 2-O-sulphates are not strictly essential.
On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase
Authors: KLIMACEK Mario , KAVANAGH Kathryn L. , WILSON David K. , NIDETZKY Bernd
Published: October 2003

X-ray structure of the Pseudomonas fluorescens mannitol 2-dehydrogenase ternary complex with NAD+ and d-mannitol suggests that Lys-295 provides catalytic base assistance to secondary alcohol group oxidation. We have replaced Lys-295 by site-directed mutagenesis with alanine or methionine and evaluated the catalytic significance of side-chain substitution by kinetic analysis of restoration of activity with external amines, and from pH and solvent isotope effects on the reaction catalysed by K295A (Lys-295→Ala mutant). K295A and K295M (Lys-295→Met mutants) show 3×104- and 2×106-fold lower turnover numbers respectively for d-mannitol oxidation (kcatO) at pH 10.0 than the wild-type. The second-order rate constant for non-covalent rescue of activity (kB) by free methylamine base is 31 M−1·s−1 for K295A, but only 0.021 M−1·s−1 for K295M. A Brønsted relationship of log kB (corrected for molecular size effects) and pKa of the external amine is linear (slope β=0.66±0.16; r2=0.99) for K295A-catalysed d-mannitol oxidation at pH 10.0. The kcatO values of K295A in H2O and 2H2O are linearly dependent on [OL−] in the pL range 7.5–10.5 (where L is 1H or 2H). The solvent isotope effect on kcatO is 0.69. The time course of d-fructose reduction by K295A at pH 8.2 displays a pre-steady-state burst of NADH consumption. These data support a mechanism in which the ε-NH2 group of Lys-295 participates in an obligatory pH-dependent, pre-catalytic equilibrium which may control alcohol/alkoxide equilibration of enzyme-bound d-mannitol and activates the C2 atom for subsequent catalytic oxidation by NAD+.
Post-translational incorporation of the antiproliferative agent azatyrosine into the C-terminus of α-tubulin
Authors: PURRO Silvia A. , BISIG C. Gastón , CONTIN María A. , BARRA Héctor S. , ARCE Carlos A.
Published: October 2003

Detyrosination/tyrosination of tubulin is a post-translational modification that occurs at the C-terminus of the α-subunit, giving rise to microtubules rich in either tyrosinated or detyrosinated tubulin which coexist in the cell. We hereby report that the tyrosine analogue, azatyrosine, can be incorporated into the C-terminus of α-tubulin instead of tyrosine. Azatyrosine is structurally identical to tyrosine except that a nitrogen atom replaces carbon-2 of the phenolic group. Azatyrosine competitively excluded incorporation of [14C]tyrosine into tubulin of soluble brain extract. A newly developed rabbit antibody specific to C-terminal azatyrosine was used to study incorporation of azatyrosine in cultured cells. When added to the culture medium (Ham's F12K), azatyrosine was incorporated into tubulin of glioma-derived C6 cells. This incorporation was reversible, i.e. after withdrawal of azatyrosine, tubulin lost azatyrosine and reincorporated tyrosine. Azatyrosinated tubulin self-assembled into microtubules to a similar degree as total tubulin both in vitro and in vivo. Studies by other groups have shown that treatment of certain types of cultured cancer cells with azatyrosine leads to reversion of phenotype to normal, and that administration of azatyrosine into animals harbouring human proto-oncogenic c-Ha-ras prevents tumour formation. These interesting observations led us to study this phenomenon in relation to tubulin status. Under conditions in which tubulin was mostly azatyrosinated, C6 cells remained viable but did not proliferate. After 7–10 days under these conditions, morphology changed from a fused, elongated shape to a rounded soma with thin processes. Incorporation of azatyrosine into the C-terminus of α-tubulin is proposed as one possible cause of reversion of the malignant phenotype.
Human salivary MUC7 mucin peptides: effect of size, charge and cysteine residues on antifungal activity
Authors: SITU Hongsa , WEI Guoxian , SMITH Christina J. , MASHHOON Shirin , BOBEK Libuse A.
Published: October 2003

We have previously shown that MUC7 (human salivary low-molecular-mass mucin) 20-mer: LAHQKPFIRKSYKCLHKRCR (residues 32–51 of the parent MUC7, with a net positive charge of 7) possesses a broad-spectrum antimicrobial activity [Bobek and Situ (2003) Antimicrob. Agents Chemother. 47, 645–652]. The aims of the present study were to determine the minimum peptide chain length and its location within the 20-mer region that retains potent antifungal activity against Candida albicans and Cryptococcus neoformans and to examine the effect of net charge of the peptide as well as the role of cysteine residues on the fungicidal activity. First, several C-terminal truncated MUC7 20-mer fragments (16-mer, 12-mer, 11-mer, 10-mer and 8-mer) and one N-terminal fragment (8-mer-N) were synthesized and tested. The results showed that MUC7 12-mer, located at the C-terminal region of MUC7 20-mer, having a net charge of +6 and exhibiting an amphipathic helical conformation, not only retained but exceeded the antifungal activity of that of 20-mer. Secondly, several variants of the 12-mer peptide containing a lower or no net positive charge, or no cysteine residues were synthesized and tested. A clear correlation between the net positive charge of the 12-mer, its potency and initial interaction of peptide with fungal cells was found by killing assays, fluorescence microscopy and fungal cell-membrane potential measurements. Furthermore, cysteine residues, which play a critical role in bacterial binding, were found to be not important for the fungicidal activity of these peptides. These results identified MUC7 12-mer as a potential candidate for development into a novel antifungal therapeutic agent.
Wortmannin alters the intracellular trafficking of the bradykinin B2 receptor: role of phosphoinositide 3-kinase and Rab5
Authors: HOULE Steeve , MARCEAU François
Published: October 2003

Wortmannin reportedly induces the formation of enlarged cytoplasmic endosomes. Such vesicles were observed in a definite time window after wortmannin treatment (250 nM) in HEK-293 cells stably expressing a B2R (B2 receptor)–green fluorescent protein conjugate and other cell types. The alternative PI3K (phosphoinositide 3-kinase) inhibitor LY 294002 (100 μM) and a dominant-negative form of the enzyme (p85α ΔiSH2) induce a more modest vesicle enlargement. PI3K inhibition by drugs did not affect agonist-induced [3H]arachidonate release. The wortmannin-induced formation of giant endosomes also involves Rab5 activity, since a dominant-negative form of this GTPase (Rab5 S34N) partially inhibits the wortmannin effect and a constitutively active form of Rab5 (Rab5 Q79L) induces the formation of enlarged endosomes. Moreover, agonist stimulation targeted B2R–green fluorescent protein towards the periphery of the giant vesicles and led to partial receptor degradation only in wortmannin-treated cells. Receptor degradation was decreased by protease inhibitors and by bafilomycin A1, a drug that inhibits lysosome function. Accumulation of fluorescent material inside the enlarged endosomes was observed in cells treated with bafilomycin A1, wortmannin and an agonist. [3H]Bradykinin binding was decreased in HEK-293 cells treated with both wortmannin and the agonist, but not with either separately. Furthermore, a wortmannin-induced functional down-regulation of B2R was observed in rabbit jugular veins after repeated agonist stimulation (contractility assay). This is the first report of a G-protein-coupled receptor down-regulation induced by an alteration of its usual routing in the cell. These results suggest that both PI3K and Rab5 influence B2R intracellular trafficking.
Identification of a novel Na+- and Cl−-coupled transport system for endogenous opioid peptides in retinal pigment epithelium and induction of the transport system by HIV-1 Tat
Authors: HU Huankai , MIYAUCHI Seiji , BRIDGES Christy C. , SMITH Sylvia B. , GANAPATHY Vadivel
Published: October 2003

The endogenous opioid peptides enkephalins, dynorphins and endorphins consist of five or more amino acids. These peptides participate in a multitude of biological functions in mammalian cells by interacting with different subtypes of opiate receptors located on the plasma membrane and in the nucleus. Here we report on the identification of a new peptide transport system in the human retinal pigment epithelial (RPE) cells that transports a variety of endogenous opioid peptides with high affinity. We identified this novel, hitherto unrecognized, transport system when we were analysing the differential effects of Tat, the transacting factor encoded by HIV-1, on various transport processes in RPE cells. This transport system is markedly induced by Tat. This opioid transport system is energized by transmembrane Na+ and Cl− gradients and is distinct from any of the previously identified transport systems for opioid peptides in mammalian cells. Free amino acids, dipeptides, tripeptides and non-peptide opiate receptor antagonists are excluded by this newly identified transport system. The affinities of endogenous opioid peptides for this system are in the range of 0.4–40 μM. The identification of the high-affinity Na+- and Cl−-coupled transport system in mammalian cells that is specific for endogenous opioid peptides and is induced by HIV-1 Tat is of significance not only to the biology of opioid peptides but also to the pathology of HIV-1 infection in humans.