bluuebunny/crossref_metadata_2025 huggingface dataset:
Both type I and type II interferons down‐regulate human tumor necrosis factor receptors in human hepatocellular carcinoma cell line Hep G2
Authors: Aggarwal Bharat B. , Pandita Raj
Published: July 2002

It is well known that Interferon‐γ (IFN‐γ type II) potentiates various responses of human tumor necrosis factor (TNF) in a wide variety of cells and that this potentiation is accompanied by the up‐regulation of TNF receptor synthesis. In the present studies we examined the regulation of TNF receptors by type I and type II IFNs in a hepatocellular carcinoma cell line, HEP G2. Exposure of these cells to IFN‐γ led to a decrease in TNF receptor number (4029 vs. 2719 sites/cell) without any change in the receptor affinity (0.96 nM vs. 1.1 nM). The effect was time and dose‐dependent. Like IFN‐γ, IFN‐α and IFN‐β (type I) down‐modulated the TNF receptors on these cells. The effect of IFNs on the TNF receptors was inhibited by staurosporin, a protein kinase C (PK‐C) inhibitor. Furthermore, by the use of receptor‐specific antibodies, we found that the IFN‐dependent decrease was primarily due to the p60 form of the TNF receptor. Our results presented are the first to demonstrate that IFNs can also down‐modulate TNF receptors in certain cells and that this effect is mediated through PK‐C.
Amylin and epinephrine have no direct effect on glucose transport in isolated rat soleus muscle
Authors: Pittner Richard A. , Wolfe-Lopez Deborah , Young Andrew A. , Rink Timothy J.
Published: July 2002

Amylin and epinephrine did not significantly affect insulin stimulated, or basal, 3‐O‐methylglucose transport in isolated rat soleus muscle, as measured by the release of 3‐O‐methylglucose from pre‐loaded tissue. Both amylin and epinephrine inhibited insulin‐stimulated 2‐deoxyglucose uptake (by 25% and 38%, respectively) in soleus muscle from fed rats but not from fasted rats. The latter results are consistent with amylin and epinephrine stimulating glycogenolysis and inhibiting hexokinase activity by intracellular accumulation of glucose 6‐phosphate. We conclude that amylin, like epinephrine, does not specifically inhibit glucose transporters in skeletal muscle.
In vitro characterization of the ORF1‐ntrBC promoter of Rhizobium etli
Authors: Martino Matilde , Riccio Anna , Defez Roberto , Iaccarino Maurizio , Patriarca Eduardo J.
Published: July 2002

Rhizobium sigma vegetative‐dependent promoters are different from those of enteric bacteria and have never been characterized before. We report here the biochemical characterization of the ORFI‐ntrBC promoter of Rhizobium etli. The minimal promoter region was located by means of a transcriptional fusion and further characterized by in vitro transcription and gel retardation experiments. Oligonucleotides used as DNA competitors in runoff transcription experiments allowed the precise localisation of the promoter region. Protein extracts from an ntrC
+, but not from an ntrC
− strain, inhibited in vitro transcription. The NtrC protein was found to bind specifically to the promoter, where an NtrC binding site overlapping the transcription initiation site, is present.
Donor‐side photoinhibition in photosystem II from Chlamydomonas reinhardtii upon mutation of tyrosine‐Z in the D1 polypeptide to phenylalanine
Authors: Minagawaa Jun , Kramer David M. , Kanazawa Atsuko , Crofts Antony R.
Published: July 2002

When tyrosine‐Z of the D1‐polypeptide of the photosystem II from Chlamydomonas reinhardtii was changed to phenylalanine, the rapid donor to P680+ was lost, and P680+ accumulated on illumination. The rapid donation from tyrosine‐Z was replaced by a slow electron transfer from an endogenous donor. Spectrophotometric measurements showed that carotenoids and chlorophylls were bleached by the P680
+ either directly or indirectly upon illumination. The carotenoid bleaching was inhibited in the presence of SOD or catalase, but the reaction did not require molecular oxygen as an electron acceptor. These observations led us to conclude that active oxygen radicals, possibly hydroxyl radicals, take part in the destruction of carotenoids in the Y161F mutant. Possible mechanisms for the destruction are discussed.
Immunocytochemical study of lampbrush chromosomes of the urodele Pleurodeles waltl: Axial granules are recognized by the mitosis‐specific monoclonal antibody MPM‐2
Authors: Pyne Chandra K. , Loones Marie‐Thérèse , Simon Françoise , Zhou Zi Jian
Published: July 2002

Summary— The lampbrush chromosomes of the urodele Pleurodeles waltl have been studied using the mitosis‐specific monoclonal antibody MPM‐2. Immunofluorescence studies revealed that MPM‐2 stains structures associated with axial granules, numerous other chromomeres, telomeres and certain chiasmata. These structures showed a negative reaction with the anti‐DNA monoclonal antibody AC‐30‐10. In course of meiotic condensation of the chromosomes, in growing and maturating oocytes, the number of such structures associated with the chromosome axis was found to diminish progressively. These granular structures have been found to be formed by fine fibrils about 5 nm in diameter. Immunogold labeling confirmed the results of immunofluorescence studies. MPM‐2 was also found to stain two other types of structures observed in association with the lampbrush chromosome axis in P waltl, viz the sphere organelle (only in later stages of oogenesis) and the structure known as ‘M’ which is singular to this material.
Progressive development of gap junctions during growth of human pancreatic duct cells (Capan‐1) in vitro and in vivo
Authors: Monzat Véronique , Fanjul Marjorie , Leclerc Catherine , Palévody Claude , Briand Jean‐Paul , Jarry‐Guichard Thérèse , Gabrion Jacqueline , Moreau Marc , Hollande Etienne
Published: July 2002

Summary— Among their numerous functions, gap junctions play a crucial role in proliferation, differentiation and secretion processes, although their existence and potential role in ion secretion in human pancreatic ducts have yet to be established. To investigate the morphogenesis and the role of gap junctions in human pancreatic duct cells, the Capan‐1 cell line maintained in culture or heterotransplanted into nude mice was employed as model system. Capan‐1 cells polarize during their growth in vivo and in vitro forming duct‐like structures. Furthermore in culture, after confluence, these cells form domes, which is indicative of ion exchange processes. After treatment with tannic acid and freeze‐fracture, gap junctions were observed along the basolateral membranes of Capan‐1 cells on electron microscopic examination. The presence of alkaline phosphatases on gap junctions was demonstrated cytoenzymatically. In addition, cell‐to‐cell communication was visualized by microinjection of Lucifer yellow. During differentiation of Capan‐1 cells in culture, the frequency of intercellular communications increased markedly over the period (days 11–13) when the cells form duct‐like structures. The increase in gap junctions was demonstrated by analysis of the polarized cells organized in duct‐like structures that are commonly observed in the tumors formed by heterotransplantation of Capan‐1 cells into nude mice. Furthermore, gap junctions associated with tight junctions were also observed in the cells forming such structures. The role of gap junctions in ion exchange was evaluated by counting the number of domes in cultures treated with heptanol. Heptanol (an uncoupling agent of gap junction communication) completely inhibited dome formation in a reversible way, and reduced the frequency of intercellular communications by 44%. These results suggest that the gap junctions expressed by Capan‐1 cells are involved in ion secretion by the human cancerous pancreatic duct cell line, Capan‐1. In the present study, we show that: i) the expression of gap junctions is linked to development of the spatial conformation of the cells; and ii) gap junctions may be involved in ion secretion.
The initial pathogenesis of cadmium induced renal toxicity
Authors: Griffin Julian L. , Walker Lee A. , Troke Jeff , Osborn Dan , Shore Richard F. , Nicholson Jeremy K.
Published: July 2002

The novel application of magic angle spinning 1H NMR spectroscopy, coupled with pattern recognition techniques, has identified biochemical changes in lipid and glutamate metabolism that precede classical nephrotoxicity. These changes occurred in the bank vole (Clethrionomys glareolus) after chronic dosing, at a low level of exposure and at a renal Cd2+ concentration (8.4 μg/g dry wt) that was nearly two orders of magnitude below the WHO critical organ concentration (200 μg/g wet wt). These early stage effects of Cd2+ on the biochemistry of renal tissue may reflect adaptation mechanisms to the toxic insult or the preliminary stages of the toxicological cascade.
Mechanism for the peroxynitrite scavenging activity by anthocyanins
Authors: Tsuda Takanori , Kato Yoji , Osawa Toshihiko
Published: July 2002

We show that anthocyanins can function as potent inhibitors of the formation of nitrated tyrosine in vitro, and clarified how pelargonidin (Pel), which has a mono‐hydroxyl group on the B‐ring, can scavenge peroxynitrite (ONOO−) by detection of the reaction products. Pel was reacted with ONOO−, then the reaction mixture was analyzed using high‐performance liquid chromatography (HPLC). The HPLC analyses showed two novel peaks assumed to be the reaction products. Based on the instrumental analyses, the reaction products were identified as p‐hydroxybenzoic acid and 4‐hydroxy‐3‐nitrobenzoic acid. Pel can protect tyrosine from undergoing nitration through the formation of p‐hydroxybenzoic acid and 4‐hydroxy‐3‐nitrobenzoic acid.
Matrix Gla protein is differentially expressed during the deposition of a calcified matrix by vascular pericytes
Authors: Canfield Ann E. , Doherty Mary Jo , Kelly Victoria , Newman Bill , Farrington Claire , Grant Michael E. , Boot-Handford Raymond P.
Published: July 2002

PCR‐based subtractive hybridisation was used to identify genes up‐regulated when pericytes undergo osteogenic differentiation and deposit a calcified matrix. cDNA pools were generated from confluent pericytes and from pericyte cultures containing calcified nodules. A pericyte cDNA library was screened with the product of the subtraction procedure (calcified minus confluent cDNA) and the majority of the positive clones were identified as matrix Gla protein (MGP). Northern analysis and immunohistochemistry demonstrated that MGP was only expressed by pericytes in calcified nodules. Antibodies to MGP inhibited the deposition of a calcified matrix by pericytes, suggesting that MGP regulates both cell differentiation and calcification.
Ras‐dependent cell cycle commitment during G2 phase
Authors: Hitomi Masahiro , Stacey Dennis W.
Published: July 2002

Synchronization used to study cell cycle progression may change the characteristics of rapidly proliferating cells. By combining time‐lapse, quantitative fluorescent microscopy and microinjection, we have established a method to analyze the cell cycle progression of individual cells without synchronization. This new approach revealed that rapidly growing NIH3T3 cells make a Ras‐dependent commitment for completion of the next cell cycle while they are in G2 phase of the preceding cell cycle. Thus, Ras activity during G2 phase induces cyclin D1 expression. This expression continues through the next G1 phase even in the absence of Ras activity, and drives cells into S phase.
The multiple origins of cooperativity in binding to multi‐site lattices
Authors: Sackett Dan L. , Saroff Harry A.
Published: July 2002

Binding events involving the reversible association of ligands with polymeric lattices of binding sites are common in biology and frequently exhibit significant cooperativity in binding. Positive and negative cooperativity in binding may be detected by characteristic changes in binding curves for multiple binding, compared to the binding expected for simple, independent binding events that are based on combinatorial considerations only. Cooperativity arises from ligand‐dependent interactions distinct from binding per se. Ligand‐dependent nearest neighbor interactions may be of two types referred to as ligand‐lattice (which can only occur if a bound ligand is unneighbored) and ligand‐ligand (which can occur if two or more bound ligands are adjacent). The molecular mechanisms underlying these two sources of cooperativity are not the same. Identical cooperative binding curves may be produced by changes from unity in parameters representing either one or both of these interaction types. Positive cooperativity may equally result from destabilizing ligand‐lattice interactions that disfavor initial, unneighbored binding, stabilizing ligand‐ligand interactions that favor subsequent, neighbored binding, or both. The structural origins of these are different, and cooperativity may emerge from multiple structural interactions.
Troglitazone inhibits angiotensin II‐induced DNA synthesis and migration in vascular smooth muscle cells
Authors: Graf Kristof , Xi Xiao-Ping , Hsueh Willa A , Law Ronald E
Published: July 2002

Angiotensin II (AII) plays a crucial role in controlling the proliferation and migration of vascular smooth muscle cells (VSMCs). The present study was undertaken to determine if troglitazone (Tro) has an effect on the G‐protein coupled signaling through AII type I (AT‐1) receptors in cultured rat aortic VSMCs. AII‐induced MAP kinase activation was inhibited 67.9% by Tro. AII‐induced DNA synthesis and migration was completely inhibited by Tro or by the AT‐1 receptor blocker irbesartan. The present study demonstrates that troglitazone inhibits AII‐induced DNA synthesis, migration and MAP kinase activation in VSMCs which are important molecular events for the development of neointimal hyperplasia and atherosclerosis.
Sterol 22‐desaturase, cytochrome P45061, possesses activity in xenobiotic metabolism
Authors: Kelly Steven L. , Lamb David C. , Kelly Diane E.
Published: July 2002

CYP61 was revealed in the sequencing of the yeast genome on chromosome XIII and was the last member of the CYP superfamily in yeast to be discovered. We show here that besides the housekeeping role in 22‐desaturation during ergosterol biosynthesis the enzyme is also that responsible for benzo(a)pyrene metabolism/promutagen activation by yeast in genotoxicity assays. This enzyme may represent an ancestral activity for the superfamily which allowed xenobiotic metabolism for the first time.
Cell cycle dependent toxicity of an amphiphilic synthetic peptide
Authors: Pellegrin Pierre , Menard Claudine , Mery Jean , Lory Philippe , Charnet Pierre , Bennes René
Published: July 2002

The cytotoxic properties of an amphiphilic synthetic peptide are presented. Comparative analysis of proliferating, differentiated and confluent H9C2 adherent cells and L1210 cells in suspension shows a correlation between toxicity and cell stage (proliferating cells). Electrophysiological measurements on Xenopus laevis oocytes bathed in the peptide also demonstrated the induction of cationic currents, which is voltage and phosphate dependent. These results allow us to hypothesize that the observed toxicity is related to membrane hyperpolarization of proliferating cells at the G1/S cell cycle phase transition.
The Ca2+‐induced pore opening in mitochondria energized by succinate‐ferricyanide electron transport
Authors: Krasnikov Boris F , Kuzminova Alevtina E , Zorov Dmitry B
Published: July 2002

The oxygen dependence of the mitochondria permeability transition pore was under study in non‐respiring rat liver mitochondria. Oxygen in the medium was depleted by saturation of the incubation medium with N2 and spontaneously by mitochondrial respiration followed by the addition of glucose/glucose oxidase. After the anaerobic state had been reached, ferricyanide has been added to support succinate‐driven energization in the absence of oxygen. In the other set of the experiments KCN was added to block operation of the respiratory chain under aerobic conditions. Again, ferricyanide was added as an electron acceptor. Superoxide dismutase was added to trap superoxide anion radicals. Under either hypoxic conditions or in the presence of cyanide, calcium ions were shown to induce the permeability transition. The concentration of Ca2+ required was lower than under conditions of active respiration. In both cases, the transition was prevented by cyclosporine A.
Anandamide amidohydrolase reacting with 2‐arachidonoylglycerol, another cannabinoid receptor ligand
Authors: Goparaju Sravan Kumar , Ueda Natsuo , Yamaguchi Hiroko , Yamamoto Shozo
Published: July 2002

Two endogenous ligands for cannabinoid receptors, anandamide (arachidonylethanolamide) and 2‐arachidonoylglycerol, lose their biological activities by enzymatic hydrolysis. A cDNA for a rat liver enzyme hydrolyzing anandamide as well as oleamide was overexpressed in COS‐7 cells. When the particulate fraction was allowed to react with 2‐arachidonoylglycerol, arachidonic acid was produced. In contrast, this hydrolytic reaction did not occur with the control cells. The hydrolysis of 2‐arachidonoylglycerol proceeded about 4‐fold faster than the anandamide hydrolysis with a K
m value as low as 6 μM and an optimal pH of 10. Phenylmethylsulfonyl fluoride and methyl arachidonyl fluorophosphonate inhibited the hydrolysis of both anandamide and 2‐arachidonoylglycerol in parallel. Furthermore, the hydrolysis of [14C]2‐arachidonoylglycerol was inhibited by anandamide dose‐dependently. These results suggest that anandamide and 2‐arachidonoylglycerol can be inactivated by the same enzyme.
Effect of inhibitors of signal transduction on IGF‐1‐induced protein synthesis associated with hypertrophy in cultured neonatal rat ventricular myocytes
Authors: Lavandero Sergio , Foncea Rocı́o , Pérez Viviana , Sapag-Hagar Mario
Published: July 2002

IGF‐1 increased 2‐fold protein synthesis in cardiac myocytes. Genistein, whether added during preincubation or with IGF‐1 at the start of incubation, significantly inhibited the IGF‐1‐induced stimulation of protein synthesis, autophosphorylation of the β‐subunit of IGF‐1 receptor and inhibition of ERK. When added 1 or 6 h after IGF‐1, however, genistein was without effect. IGF‐1‐stimulated protein synthesis was also significantly inhibited by PD‐098059, staurosporine, and rapamycin, but not by wortmannin, in cardiac myocytes. Some inhibitors produced a reduction in cell size. Activation of the ERK cascade by IGF‐1 may be responsible for some of the features associated with cardiac myocyte hypertrophy.
G1 phase arrest by the phosphatidylinositol 3‐kinase inhibitor LY 294002 is correlated to up‐regulation of p27Kip1 and inhibition of G1 CDKs in choroidal melanoma cells
Authors: Casagrande Fabrice , Bacqueville Daniel , Pillaire Marie-Jeanne , Malecaze François , Manenti Stéphane , Breton-Douillon Monique , Darbon Jean-Marie
Published: July 2002

We have investigated the effect of the flavonoid derivative LY 294002, a potent and selective phosphatidylinositol 3‐kinase inhibitor, on cell cycle progression in human choroidal melanoma cells. We demonstrate that LY 294002 induces a specific G1 block in asynchronously growing cells leading to an almost complete inhibition of cell proliferation after three days of treatment. When melanoma cells are released from a nocodazole‐induced G2/M block, LY 294002 is shown to delay and greatly restrain the G1/S transition. The inhibitor is able to exert its action as long as it is added during the G1 progression and before the cells enter in S phase. We report that the LY 294002‐induced G1 arrest is closely correlated to inhibition of CDK4 and CDK2 activities leading to the impairment of pRb phosphorylation which normally occurs during G1 progression. While the inhibition of CDK4 may be attributed at least in part to the decline in CDK4 protein level, CDK2 activity reduction is rather due to the up‐regulation of the CDK inhibitor p27Kip1 and to its increased association to CDK2.
Xanthine oxidoreductase is asymmetrically localised on the outer surface of human endothelial and epithelial cells in culture
Authors: Rouquette Magali , Page Susanna , Bryant Richard , Benboubetra Mustapha , Stevens Cliff R , Blake David R , Whish William D , Harrison Roger , Tosh David
Published: July 2002

Subcellular localisation of xanthine oxidoreductase (XOR) was determined by indirect immunofluorescence using confocal microscopy in human endothelial and epithelial cell lines and in primary cultures of human umbilical vein endothelial cells. XOR was diffusely distributed throughout the cytoplasm but with higher intensity in the perinuclear region. In non‐permeabilised cells, XOR was clearly seen to be asymmetrically located on the outer surfaces, showing, in many cases, a higher intensity on those faces apposed by closely neighbouring cells. Such specific distribution suggests a functional role for the enzyme in cell‐cell interactions, possibly involving signalling via reactive oxygen species
Homologous p35 proteins of baculoviruses show distinctive anti‐apoptotic activities which correlate with the apoptosis‐inducing activity of each virus1
Authors: Morishima Nobuhiro , Okano Kazuhiro , Shibata Takehiko , Maeda Susumu
Published: July 2002

The anti‐apoptotic activity of p35s from two baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV) and Bombyx mori NPV (BmNPV), was compared in mammalian cells. AcNPV p35 efficiently blocked apoptosis induced by caspase overexpression, but BmNPV p35 did so very poorly. Analysis of chimeric p35s and in vitro cleavage of wild type p35s suggest that the cleavage efficiency of p35 correlates with the blocking activity. Single amino acid substitutions of BmNPV p35 with those observed in AcNPV p35, however, resulted in significant loss of its anti‐apoptotic activity. We speculate that sequences flanking the cleavage site have uniquely evolved during baculovirus evolution.
Application of polymannosylated cystatin to surimi from roe‐herring to prevent gel weakening
Authors: Nakamura Soichiro , Ogawa Masahiro , Saito Masayoshi , Nakai Shuryo
Published: July 2002

A recombinant glycosylated cystatin with a polymannosyl chain was added to roe‐herring surimi for preventing gel weakening due to autolysis during cooking. Proteolysis of myosin heavy chain in the surimi was effectively suppressed while cooking at 90°C for 20 min after preincubation at 40°C for 30 min. The glycosylation of cystatin improved the stability against heating as well as proteolysis by cathepsin D. This process markedly improved the texture of the cooked surimi gel with gel strength 2.5 times that of unglycosylated control cystatin.
Membrane‐type 1 MMP (MMP‐14) cleaves at three sites in the aggrecan interglobular domain
Authors: Fosang Amanda J , Last Karena , Fujii Yutaka , Seiki Motoharu , Okada Yasunori
Published: July 2002

An aggrecan G1‐G2 substrate was used to determine sites within the interglobular domain that were susceptible to cleavage by MT1‐MMP. Degradation products were identified by Western blotting with neo‐epitope antibodies specific for MMP‐derived N‐ and C‐terminal sequences. The results showed that MT1‐MMP cleaved at the N341‐F342 and D441‐L442 bonds, as shown for other MMPs, and also at a site 13 amino acids C‐terminal to the N341‐F342 site. The G2 product of this additional cleavage was identified by sequence analysis and revealed an N‐terminus commencing T355VxxPDVELPLP. The data are consistent with MT1‐MMP cleavage at three sites in the aggrecan interglobular domain; one at N342‐F342, a second at D441‐L442 and a third at Q354‐T355.
Cloning and characterization of a novel human inwardly rectifying potassium channel predominantly expressed in small intestine
Authors: Partiseti Michel , Collura Vincent , Agnel Magali , Culouscou Jean-Michel , Graham David
Published: July 2002

A new member of the two transmembrane domain potassium (K+) channel family was identified and isolated from a human brain cDNA library. The cDNA clone contains an open reading frame which encodes a 360 amino acid sequence with a characteristic P domain flanked by two hydrophobic regions representing the membrane spanning segments. The closest homologue of this gene product is the inwardly rectifying potassium channel subunit, Kir1.2 (identity approximately 42%). Northern blot analysis of human tissues with a selective cDNA probe for this new K+ subunit showed a single major transcript of 3.4 kb predominantly expressed at high levels in small intestine, with lower levels in stomach, kidney and brain. The main regions of expression in the central nervous system were medulla, hippocampus and corpus callosum. cRNA‐injected oocytes and transiently transfected HEK293 cells expressed a K+ conductance which displays an inward rectification. This conductance is blocked by cesium and barium but is insensitive to tolbutamide and diazoxide even upon co‐transfection of this novel subunit with the plasmid encoding the sulfonylurea receptor SUR1. Taken together, these results demonstrate that we have isolated and characterized a novel K+ channel subunit belonging to the inwardly rectifying K+ (Kir) channel family to which, upon homology classification, we have given the nomenclature Kir7.1.
Inhibitory effect of acidic pH on OmpC porin: wild‐type and mutant studies
Authors: Liu Nazhen , Delcour Anne H
Published: July 2002

By use of the patch clamp technique, we have compared the electrophysiological signature of OmpC porin channels at neutral and acidic pH. The perfusion of pH 5.4 buffer to the periplasmic side of excised patches promoted the closure or block of ∼20% of the open porins present in the patch without changes in their single channel conductance. Besides this effect on the main, long‐lived open state, lowering the pH also suppressed the spontaneous transitions of channels to another distinct short‐lived open state. The inhibitory effect on the opening kinetics was particularly visible in two mutants (K16Q and E109Q) in which transitions to the short‐lived open state are enhanced by the mutations themselves at pH 7.2. On the other hand, the R124Q mutant responded to acidic pH by an increased gating to the short‐lived open state. The results suggest that acidic pH stabilizes a closed state of OmpC porin, and that the pH sensitivity might be conferred in part by R124, but not by K16 or E109.
An innovative sandwich ELISA system based on an antibody cocktail for gluten analysis
Authors: Sorell Luis , López Juan Antonio , Valdés Israel , Alfonso Patricia , Camafeita Emilio , Acevedo Boris , Chirdo Fernando , Gavilondo Jorge , Méndez Enrique
Published: July 2002

A cocktail sandwich ELISA based on the employ of two monoclonal antibodies (MAbs) as coating antibodies and a third MAb conjugated to horseradish peroxidase has been developed for the analysis of gluten in foods. Given that each MAb displays a wide specificity spectrum for wheat, barley, rye and oats prolamins, their combination for ELISA ensures a high crossreactivity with most of the potentially toxic gliadin, hordein, secalin and avenin protein family. One of the unprecedented features of the cocktail sandwich ELISA is that it permits for the first time analysis of barley hordeins in foods, which is unattainable using conventional or commercial ELISA kits. Besides, gliadins, hordeins and secalins are recognised to the same extent. The system provides a high detection sensitivity for gliadins, hordeins, secalins and avenins (1.5, 0.05, 0.15 and 12 ng/ml, respectively). The working linear range comprises 3–100 ng/ml with a gliadin detection limit of 1.5 ppm. This limit of detection is even better than that demanded in the latest Codex recommendation, 10 ppm. Cocktail ELISA data were contrasted with those of commercial ELISA kits and confirmed by mass spectrometry, a non‐immunological technique which provides evidence for the occurrence of false positive results with the commercial kits.
Dephosphorylation of distinct sites on the 20 kDa myosin light chain by smooth muscle myosin phosphatase
Authors: Feng Jianhua , Ito Masaaki , Nishikawa Masakatsu , Okinaka Tsutomu , Isaka Naoki , Hartshorne David J , Nakano Takeshi
Published: July 2002

The dephosphorylation of the myosin light chain kinase and protein kinase C sites on the 20 kDa myosin light chain by myosin phosphatase was investigated. The myosin phosphatase holoenzyme and catalytic subunit, dephosphorylated Ser‐19, Thr‐18 and Thr‐9, but not Ser‐1/Ser‐2. The role of non‐catalytic subunits in myosin phosphatase was to activate the phosphatase activity. For Ser‐19 and Thr‐18, this was due to a decrease in K
m and an increase in k
cat and for Thr‐9 to a decrease in K
m. Thus, the distinction between the various sites is a property of the catalytic subunit.
Selective activation of phospholipase D2 by unsaturated fatty acid
Authors: Kim Jae Ho , Kim Yong , Lee Sang Do , Lopez Isabel , Arnold Rebecca S. , Lambeth J.David , Suh Pann-Ghill , Ryu Sung Ho
Published: July 2002

Although oleate has been implicated in the regulation of phospholipase D (PLD) activity, the molecular identity of the oleate‐stimulated PLD is still poorly understood. We now report that oleate selectively stimulates the enzymatic activity of PLD2 but not of PLD1, with an optimal concentration of 20 μM in vitro. Intriguingly, phosphatidylinositol 4,5‐bisphosphate (PIP2) synergistically stimulates the oleate‐dependent PLD2 activity with an optimal concentration of 2.5 μM. These results provide the first evidence that oleate is a PLD2‐specific activating factor and PLD2 activity is synergistically stimulated by oleate and PIP2.
Cloning and expression of three cecropin cDNAs from a mosquito cell line
Authors: Sun Dongxu , Eccleston Eric D. , Fallon Ann M.
Published: July 2002

We have characterized full‐length cDNAs encoding three isoforms of the antibiotic cecropin secreted by the C7–10 cell line from the mosquito, Aedes albopictus. The existence of two cecropin isoforms that differed from the previously described AalCecA was predicted by mass spectrometry and amino acid sequence analysis of peptides that eluted from reversed phase high performance liquid chromatography as a single peak just behind the previously described cecropin, AalCecA. Based on the amino acid sequence of the mature AalCecA peptide, we designed primers that amplified partial cDNAs encoding three different A. albopictus cecropins in reverse transcriptase polymerase chain reactions. Rapid amplification of cDNA ends was then used to complete the cDNA sequences of AalCecA, AalCecB and AalCecC, respectively. Each cDNA encoded a translation product containing a signal peptide, a pro region, and a mature cecropin peptide consistent with amino acid sequence data from chymotryptic digests. Although the mosquito cecropins shared 70–86% identity among each other, they shared only ∼40% identity to cecropins from Drosophila melanogaster. Each of the cecropins was expressed within 2 to 4 h after induction, and transcripts measuring 0.3 to 0.5 kb continued to accumulate over 24 h. The three cecropins were secreted in roughly equimolar proportions, and 30 to 90% of AalCecB was amidated at the terminal glycine residue. In contrast, amidated forms of AalCecA and AalCecC constituted a smaller proportion of these isoforms.
TOP mRNAs are translationally inhibited by a titratable repressor in both wheat germ extract and reticulocyte lysate
Authors: Biberman Yael , Meyuhas Oded
Published: July 2002

Vertebrate TOP mRNAs contain a 5′ terminal oligopyrimidine tract (5′ TOP), which is subject to selective translational repression in non‐growing cells or in cell‐free translation systems. In the present study, we monitored in vitro the effect of increasing amounts of a 16 nucleotides long oligoribonucleotide representing the 5′ terminus of mouse ribosomal protein S16 mRNA on the translation of TOP and non‐TOP mRNAs. Our results demonstrate that the wild‐type sequence (but not its mutant counterparts) derepresses the translation of mRNAs containing 5′ TOP motifs, but failed to stimulate the translation of non‐TOP mRNAs, even if the latter differed only by a single nucleotide from their 5′ TOP‐containing counterparts. Similar results have been obtained with both wheat germ extract and rabbit reticulocyte lysate. It appears, therefore, that translational repression of TOP mRNAs is achieved in vitro by the accumulation of a titratable repressor rather than by the loss of an activator and that this repressor recognizes multiple TOP mRNAs with a diverse set of 5′ TOP motifs.
Development of a quantitative gene expression assay for Chlamydia trachomatis identified temporal expression of σ factors
Authors: Mathews Sarah A , Volp Kym M , Timms Peter
Published: July 2002

Chlamydia trachomatis is an important human pathogen which possesses a unique bi‐phasic developmental cycle. We used lightcycler methodology to quantitatively measure gene transcript levels in C. trachomatis strain L2. By measuring 16S rRNA transcript levels, we determined C. trachomatis L2 to have a generation time of approximately 3 h and an inclusion burst size of 200–300 particles. The three chlamydial σ factor genes rpoD (σ66), rpsD (σ28) and rpoN (σ54) exhibited different patterns of temporal expression. rpoD was central to early chlamydial development, whereas rpsD and rpoN were temporally expressed, coinciding with elementary body (EB) to reticulate body (RB) conversion and RB to EB conversion, respectively.
Is pantetheinase the actual identity of mouse and human vanin‐1 proteins?
Authors: Maras Bruno , Barra Donatella , Duprè Silvestro , Pitari Giuseppina
Published: July 2002

Pantetheinase is an amidohydrolase involved in the dissimilative pathway of CoA, allowing the turnover of the pantothenate moiety. We have determined the N‐terminal sequence as well as the sequences of a number of tryptic and chymotryptic peptides of the protein isolated from pig kidney. These sequence stretches were used as probes to search in the SwissProt database and significant similarities were found with a GPI‐anchored protein (mouse vanin‐1, with a suggested role in lymphocyte migration), with two putative proteins encoded by human cDNAs (VNN1 and VNN2) and with human biotinidase. On the basis of sequence similarity, we propose that vanin‐1 and VNN1 should be identified as pantetheinase.
Bone loss rate in the lumbar spine: a comparison between natural and surgically induced menopause
Authors: Yildiz Akgün , Şahin İzzet , Göl Kemal , Taner Zeki , Ulutürk Ayşe , Biberoĝlu Kutay
Published: July 2002

Objective: To compare the bone loss between natural and surgically induced menopause by dual photon absorbsiometry (DPA) and quantitative computed tomography (QCT). Methods: The study group included 365 women, who were in menopause for at least 6 months. Of all patients, 272 became menopausal naturally and 93 surgically. Spinal bone mineral density (BMD) of the women was determined by DPA in 201 patients and by QCT in 164. Regression analysis was used to correlate BMD and months since menopause. Results: Measurements with DPA did not show any significant difference in bone loss between natural and surgical menopause patients, whereas in the group of patients of whom BMD is measured by QCT, a relatively increased bone loss rate is found in surgically induced menopause patients. Conclusions: Oopherectomized women seemed to suffer a relatively higher bone loss rate compared with natural menopause.
MAP kinase dynamics in yeast
Authors: Van Drogen Frank , Peter Matthias
Published: July 2002

Summry— MAP kinase pathways play key roles in cellular responses towards extracellular signals. In several cases, the three core kinases interact with a scaffold molecule, but the function of these scaffolds is poorly understood. They have been proposed to contribute to signal specificity, signal amplification, or subcellular localization of MAP kinases. Several MAP kinases translocate to the nucleus in response to their activation, suggesting that nuclear transport may provide a regulatory mechanism. Here we describe new applications for Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP), to study dynamic translocations of MAPKs between different subcellular compartments. We have used these methods to measure the nuclear/cytoplasmic dynamics of several yeast  MAP kinases, and in particular to address the role of scaffold proteins for MAP‐kinase signaling.
Drug‐induced alterations in rat peritubular cell cytoskeleton result in proteoglycan synthesis modifications. Comparison with some intracellular signaling pathways
Authors: Thiébot Bénédicte , Bilinska Barbara , Langris Monique , Bocquet Jean , Carreau Serge
Published: July 2002

The influence of phorbol myristate acetate (PMA), dibutyryl cAMP and insulin‐like growth factor (IGF‐1) as well as cytoskeletal disrupting drugs on morphological changes has been studied in peritubular cells isolated from immature rat testis. Morphological studies were combined with immunofluorescence investigations of cytoskeletal elements and their rearrangements by various agents. The results were correlated with modulation of proteoglycan synthesis. Peritubular cells exposed to dibutyryl cAMP or cytochalasin D were transformed from flattened, fibroblast‐like into neuronal‐like morphology. In such cells, destruction of actin filaments was accompanied with a 50% decrease in cell‐associated proteoglycan synthesis as well as with oversulfation of total proteoglycans. On the contrary, peritubular cell shape has been slightly altered after addition of PMA, IGF‐1, vinblastine or colchicine. After these treatments, destruction or rearrangement of cytoskeletal elements was observed; cell‐layer proteoglycan synthesis remained either unchanged or increased while total proteoglycans were always undersulfated. IGF‐1, PMA and dibutyryl cAMP modified the peritubular cell morphology, cytoskeletal organization and proteoglycan production; the cytoskeleton disrupting drugs such as vinblastine, colchicine and cytochalasin D mimicked some of these effects. These observations suggest that alterations in proteoglycan biosynthesis, after activation of tyrosine kinase, protein kinase C and protein kinase A pathways might be mediated, at least in part, by the disorganization of the cytoskeleton structure.
Approaches to mass customization: configurations and empirical validation
Authors: Duray Rebecca , Ward Peter T , Milligan Glenn W , Berry William L
Published: July 2002

Mass customization is a paradox‐breaking manufacturing reality that combines the unique products of craft manufacturing with the cost‐efficient manufacturing methods of mass production. Although this phenomenon is known to exist in practice, academic research has not adequately investigated this new form of competition. In this research, we develop a configurational model for classifying mass customizers based on customer involvement in design and product modularity. We validate this typology through an empirical analysis and classification of 126 mass customizers. We also explore manufacturing systems and performance implications of the various mass customization configurations.
The development of the reticular lamina in the hamster: An examination of transitory features and their functional roles
Authors: Kaltenbach James A. , Falzarano Pamela R.
Published: July 2002

This study examines the development of the reticular lamina in the Syrian golden hamster postriatally from birth to adulthood at 2 day intervals using the scanning electron microscope. During this period, numerous transitory features emerged whose roles were concerned primarily with the development of the tectorial membrane (TM). The principal findings were as follows. (1) The surface of the developing organ of Corti produced all the fibrous material composing the minor tectorial membrane (mTM) including radial and longitudinal fiber bundles which formed the skeleton of the TM, and spongy, amorphous material which formed its intervening ground substance. (2) Throughout most of the cochlear spiral, radial fiber bundles were seen extending from the microvilli of supporting cells and projecting toward the major tectorial membrane (MTM). In most of the basal turn, but not in the apical turn, these radial bundles were interwoven with longitudinal fiber bundles which emerged from the surface of Hensen's cells. These findings indicate that the architecture of the TM is more complex in the basal turn than in the apex. (3) Increases in the dimensions of the reticular lamina resulted from the emergence of pillar cell headplates and growth in the diameter of hair cells and supporting cells. The emergence of pillar cell headplates was the principal factor contributing to increases in the radial dimension of the reticular lamina. This emergence was most dramatic between 10 and 12 days after birth (DAB) after the mTM completed its growth. Since the mTM appears to be bound medially to the MTM and laterally to the marginal pillars by 10 DAB, it seems likely that the growth of the reticular lamina after 10 DAB causes some stretching of the mTM both radially and longitudinally. (4) Completion of outer hair cell stereocilia growth at 8 DAB was followed by loss of supporting cell attachments of the TM (trabeculae) by 10 DAB, and coincided with the formation of marginal pillars from the third row of supporting cells. It is suggested that the formation of marginal pillars may be required for coupling of the TM to the tips of outer hair cell stereocilia and for induction of radial tension of the mTM. (5) Removal of the marginal pillar attachments occurred following completion of hair cell growth. (6) All structures on the reticular lamina appeared to have adult‐like characteristics by 20 DAB.
Weaver mutant mouse cerebellar granule cells respond normally to chronic depolarization
Authors: Bjerregaard Anette , Mogensen Helle S. , Hack Nicola , Balázs Robert , Jørgensen Ole Steen
Published: July 2002

We studied the effects of chronic K+‐induced membrane depolarization and treatment with N‐methyl‐d‐aspartate (NMDA) on cerebellar granule cells (CGCs) from weaver mutant mice and non‐weaver litter‐mates. The weaver mutation is a Gly‐to‐Set substitution in a conserved region of the Girk2 G prote‐incoupled inward rectifying potassium channel [Patil N., Cox D. R., Bhat D., Faham M., Myers R. M. and Peterson A. S. (1995) Nature Genet.11, 126–129] which induces early death of CGCs. The biochemical differentiation of CGCs was estimated as the rate of 2‐deoxy‐d‐glucose accumulation and the expression of neural cell adhesion molecule (NCAM). High (25 mM) K+ ion concentration or treatment with NMDA greatly promoted the biochemical differentiation of both weaver mutant and non‐weaver litter‐mate mouse CGCs. In contrast to the marked effect on biochemical differentiation in both weaver and non‐weaver mice CGSs, chronic high K+ treatment only had limited effect on survival. The survival of weaver mutant mouse CGCs in medium containing 5 mM K+ ions was very low, only 20% of the plated cells surviving at 7 days after plating, as opposed to the 50% for non‐weaver CGCs. Chronic high K+ treatment improved the relative survival of weaver mutant mouse CGCs 1.6–2.2‐fold and that of non‐weaver CGCs 1.2–1.4‐fold; the same number of CGCs (about 20% of the plated cells) were rescued by high K+ in both types of culture. The findings indicate that, in culture weaver mutant mouse, CGCs have a normal response to membrane depolarization and that the normal function of the Girk2 potassium channel is not critical for the survival of differentiated CGCs.
Sociocultural and economic aspects of quality of life measurement
Authors: Chisholm D , Bhugra D
Published: July 2002

Obtaining a level of consensus over the definition, construction and measurement of the concept of quality of life would allow for an improved degree of standardization in the assessment of clinical intervention for people with mental health problems. One of the many benefits of this standardization would be the ability to make valid and reliable comparisons between various interventions and across different groups or settings, which is of particular interest to economists. There are, however, a host of sociocultural issues that present fundamental obstacles to the satisfactory attainment of consensus over definitions and domains of quality of life. This paper considers the arguments pertinent to each of these two alternative perspectives, the economic and the sociocultural (or anthropological), and draws out the lessons that these perspectives — despite the apparent polarity that exists between them — can offer to the improved measurement of quality of life for those with mental health problems.
Microfluidic and Nanofluidic Intracellular Delivery
Authors: Hur Jeongsoo , Chung Aram J.
Published: June 2021

Innate cell function can be artificially engineered and reprogrammed by introducing biomolecules, such as DNAs, RNAs, plasmid DNAs, proteins, or nanomaterials, into the cytosol or nucleus. This process of delivering exogenous cargos into living cells is referred to as intracellular delivery. For instance, clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 gene editing begins with internalizing Cas9 protein and guide RNA into cells, and chimeric antigen receptor‐T (CAR‐T) cells are prepared by delivering CAR genes into T lymphocytes for cancer immunotherapies. To deliver external biomolecules into cells, tools, including viral vectors, and electroporation have been traditionally used; however, they are suboptimal for achieving high levels of intracellular delivery while preserving cell viability, phenotype, and function. Notably, as emerging solutions, microfluidic and nanofluidic approaches have shown remarkable potential for addressing this open challenge. This review provides an overview of recent advances in microfluidic and nanofluidic intracellular delivery strategies and discusses new opportunities and challenges for clinical applications. Furthermore, key considerations for future efforts to develop microfluidics‐ and nanofluidics‐enabled next‐generation intracellular delivery platforms are outlined.
A Computer Vision Sensor for Efficient Object Detection Under Varying Lighting Conditions
Authors: Cuhadar Can , Lau Genevieve Pui Shan , Tsao Hoi Nok
Published: June 2021

Convolutional neural networks (CNNs) have attracted much attention in recent years due to their outstanding performance in image classification. However, changes in lighting conditions can corrupt image segmentation conducted by CNN, leading to false object detection. Even though this problem can be mitigated using a more extensive CNN training set, the immense computational and energy resources required to continuously run CNNs during always‐on applications, such as surveillance or self‐navigation, pose a serious challenge for battery‐reliant mobile systems. To tackle this longstanding problem, a vision sensor capable of autonomously correcting for sudden variations in light exposure, without invoking any complex object detection software, is proposed. Such video preprocessing is efficiently achieved using photovoltaic pixels tailored to be insensitive to specific ranges of light intensity alterations. In this way, the pixels behave similarly to neurons, wherein the execution of object detection software is only triggered when light intensities shift above a certain threshold value. This proof‐of‐concept device allows for efficient fault‐tolerant object detection to be implemented with reduced training data as well as minimal energy and computational costs and demonstrates how hardware engineering can complement software algorithms to improve the overall energy efficiency of computer vision.