bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxic induction of vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability
Authors: Shima David T. , Deutsch Urban , D'Amore Patricia A.
Published: July 2002

Vessel growth is often associated with ischemia. VEGF, a potent angiogenic factor, has been shown to be induced by low oxygen concentrations. These studies were conducted to investigate the molecular basis of the hypoxia‐induced increase in VEGF mRNA. Run‐on analysis of VEGF revealed a minimal increase in the rate of gene transcription in a human retinal epithelial cell line grown under hypoxic conditions. Examination of VEGF mRNA stability revealed that the half‐life of VEGF transcripts under normoxia was short, 30–45 min, but was dramatically increased to 6–8 h in cells grown under hypoxia. Cobalt chloride, which elevates VEGF and has been suggested to be similar to hypoxia in its mechanism of action, had only a slight effect on decay rate. We postulate that hypoxia‐induced increases in mRNA stability provide the sustained increases in VEGF mRNA levels necessary to support a neovascular response.
Mouse sperm membrane potential: changes induced by Ca2+
Authors: Espinosa Felipe , Darszon Alberto
Published: July 2002

Mouse sperm resting membrane potential (E
r) (−42±8.8 mV), determined with a potential sensitive dye, depended on extracellular K+ and, in the absence of extracellular Ca2+ ([Ca2+]e), on external Na+ ([Na+]e). Ca2+ addition (>5 μM) to sperm in Ca‐free media induced a transient hyperpolarization (Ca‐ith) which strongly depended on [Na+]e and less on external Cl− ([Cl−]e). Cd2+ and Mn2+ (μM) mimicked the Ca2+ effect, but not Ba2+. The Ca‐ith was partially inhibited by ouabain (74%, IC
50 = 5.8 μM) and niflumic acid (38%, IC
50 = 240 μM), indicating the participation of the Na‐K ATPase and Cl− channels. In Ca‐free low‐Na+ media, Ca2+ addition caused a depolarization sensitive to: nimodipine (25 μM), trifluoperazine (12.5 μM) and Mg2+ (1.2 mM), suggesting the participation of Ca2+ channels. Since some inhibitors of the sperm Ca‐ith block the acrosome reaction (AR), both processes may share transport systems.
Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells
Authors: Withers Dominic J. , Coppock Hedley A. , Seufferlein Thomas , Smith David M. , Bloom Stephen R. , Rozengurt Enrique
Published: July 2002

Our results demonstrate that the novel vasoactive regulatory peptide adrenomedullin is a potent mitogen for Swiss 3T3 cells. Acting via a specific adrenomedullin receptor, it stimulates a dose‐dependent increase in DNA synthesis in synergy with insulin. Additionally, adrenomedullin stimulates further progression through the cell cycle resulting in cell proliferation, an effect that was further enhanced by the presence of insulin. Adrenomedullin rapidly induces accumulation of intracellular CAMP but does not stimulate an increase in intracellular Ca2+, activation of protein kinase C, or tyrosine phosphorylation of intracellular substrates. Adrenomedullin‐stimulated mitogenesis is markedly enhanced in Swiss 3T3 cells stably transfected with a constitutively activated Gsα, which are highly sensitive to agents that elevate cAMP, and is inhibited by the PKA inhibitor H‐89. Adrenomedullin is, thus, identified as a novel mitogenic regulatory peptide acting via cAMP.
Cell adhesion and integrin binding to recombinant human fibrillin‐1
Authors: Pfaff Martin , Reinhardt Dieter P. , Sakai Lynn Y. , Timpl Rupert
Published: July 2002

Fibrillin‐1 is a major constituent of tissue microfibrils that occur in most connective tissues, either in close association with or independent of elastin. To test possible cell‐adhesive functions of this protein, we used recombinant human fibrillin‐1 polypeptides produced in a mammalian expression system in cell attachment and solid‐phase integrin binding assays. Fibrillin‐1 polypeptides containing the single RGD sequence located in the fourth 8‐cysteine domain, mediated distinct cell adhesion of a variety of cell lines and bound to purified integrin αVβ3. Integrins αIIbβ3, α5β1, α2β1 and α1β1 did not interact with any of the recombinant fibrillin‐1 peptides. Our results indicate a novel role for fibrillin‐1 in cellular interactions mediated via an RGD motif that is appropriately exposed for recognition by integrin αVβ3.
Expression and initial characterization of recombinant mouse thrombospondin 1 and thrombospondin 3
Authors: Chen Hui , Aeschlimann Daniel , Nowlen Julie , Mosher Deane F.
Published: July 2002

To analyze the function of TSP family members, we have expressed and purified mouse TSPI and TSP3 encoded by recombinant baculoviruses in Spodoptera frugiperda cells and compared these TSPs to mouse TSP2 prepared in a similar way. Yields of purified TSP1 and TSP3 were 5–15 and 2–4 μg, respectively, per ml of conditioned medium. Mature, secreted mouse TSPI and TSP3 had the previously predicted NH2‐terminal sequences of DHVKDTSFDLFSI, and SQDLQVIDLLT, respectively. Analysis by SDS‐PAGE and rotary shadowing electron microscopy indicated that TSPI and TSP2 are disulfide bonded trimers whereas TSP3 is a disulfide‐bonded pentamer. Binding assays with 45Ca2+ as ligand and immobilized TSP1, TSP2 and TSP3 demonstrated that all three TSPs are calcium‐binding proteins. These results are consistent with previous studies of TSP structure and demonstrate that Spodoptera cells process and secrete TSPs having the same subunit organizations and structure as the native vertebrate molecules.
Different location in dark‐adapted leaves of Phaseolus vulgaris of ribulose‐1,5‐bisphosphate carboxylase/oxygenase and 2‐carboxyarabinitol 1‐phosphate
Authors: Anwaruzzaman  , Nakano Yoshihisa , Yokota Akiho
Published: July 2002

In situ RuBisCO activity was analyzed in order to determine whether 2‐carboxyarabinitol 1‐phosphate (CA1P) interacts with the enzyme in dark‐adapted leaves of Phaseolus vulgaris. Leaves ground to fine powder in liquid nitrogen were put directly into a reaction mixture containing a saturating concentration of ribulose 1,5‐bisphosphate (RuBP) to preserve the activity of RuBisCO which was in the chloroplasts. Some 70% of the total catalytic sites of RuBisCO possessed carboxylase activity in this assay, however, RuBisCO was inhibited in the absence of RuBP. CA1P seemed to be concentrated in the veins. These results indicate that RuBisCO was not complexed with CA1P in leaves.
A dnaA box can functionally substitute for the priming signals in the oriV of the broad host‐range plasmid RSF 1010
Authors: Taguchi Yoshitomo , Tanaka Katsunori , Honda Yoichi , Miao Deng-Ming , Sakai Hiroshi , Komano Tohru , Bagdasarian Michael
Published: July 2002

The initiation of replication from oriV
RSF1010, the replication origin of the broad host‐range plasmid RSF1010, depends on RepA (helicase), RepB′ (primase), and RepC (intiator protein), encoded by RSF1010 itself, while this initiation event in E. coli is independent of dnaA, dnaB, dnaC, and dnaG [Scherzinger et al. (1984) Proc. Natl. Acad. Sci. USA 81, 654–658; Scholz et al. (1985) in: Plasmids in Bacteria, pp. 243–259, Plenum, New York; Haring and Scherzinger (1989) in: Promiscuous Plasmids of Gram‐negative Bacteria, pp. 95–124, Academic Press, London; Scherzinger et al. (1991) Nucl. Acids Res. 19, 1203–1211]. We showed in this work that a newly constructed origin consisting of an oriV
RSF1010 and a DnaA protein binding site, the dnaA box, inserted near oriV
RSF1010 (oriV
RSF1010‐dnaA box) could function without RepB′ primase, but required RepA and RepC. This oriV
RSF1010‐dnaA box could not replicate in a dnaA46 strain in which only RepA and RepC were supplied, even at a permissive temperature. These results indicate that an inserted dnaA box can functionally substitute for the RSF1010‐specific ssi signals, the RepB′ dependent priming signals in oriV
RSF1010, and can direct a priming pathway different from the RSF1010‐specific one, but related to DnaA protein.
The β subunit of human rod photoreceptor cGMP‐gated cation channel is generated from a complex transcription unit
Authors: Ardell Michelle D. , Aragon Ileana , Oliveira Luanne , Porche G.Erick , Burke Emily , Pittler Steven J.
Published: July 2002

Human and bovine rod photoreceptor cGMP‐gated cation channel consists of two subunits: α (63 kDa) and β (240 kDa). The human β subunit was shown to consist partly of sequence encoded by the cDNA clone hRCNC2b. Here we present the complete sequence of the human β subunit and demonstrate that the previously reported human GAR1 gene encoding a glutamate‐rich protein (hGARP) encodes its N‐terminal portion. Using PCR, RNA blot and genomic DNA analysis, we provide evidence that the β subunit is produced from a complex locus on chromosome 16 which is also capable of generating independent transcripts corresponding to GAR1 and the C‐terminal two‐thirds of the β subunit. The results indicate that the β subunit of the cGMP‐gated cation channel is produced from an unusual locus consisting of more than one transcription unit.
Linguistic analysis of protein folding
Authors: Groß Michael
Published: July 2002

Folding of nascent chains resembles the decoding of spoken language in that information is emitted as a unidirectional, one‐dimensional string of elements, with higher structures and long‐distance interactions emerging with time. Applying a ‘pseudolinguistic’ analysis of structure to a set of all 36 possible six‐stranded antiparallel β‐sandwich topologies reveals new order principles and reduces the complexity of this family significantly. The simple connectivity diagrams (‘linguistic trees’) proposed here allow predictions of the speed and cooperativity of β‐sheet folding and help understanding the cotranslational folding from the N‐terminus.
The activity of the gluconate‐H+ symporter of Schizosaccharomyces pombe cells is down‐regulated by d‐glucose and exogenous cAMP
Authors: Caspari Thomas , Urlinger Stefanie
Published: July 2002

Schizosaccharomyces pombe cells take up d‐gluconate, as an alternative carbon source for growth, during glucose starvation or when cultured on glycerol‐containing medium. Gluconate uptake is not detectable while cells are growing logarithmically on glucose. The addition of d‐glucose as well as its non‐metabolizable analogues to glycerol‐grown cells causes an immediate loss of gluconate transport within 1 min. The reversible down‐regulation of the gluconate carrier occurs after glucose has been internalized. This regulation is triggered not only by d‐glucose but also by extracellular cAMP even in the absence of the cAMP‐dependent protein kinase (PKA1).
Massive fetomaternal hemorrhage
Authors: Tsuda H. , Matsumoto M. , Sutoh Y. , Hidaka A. , Imanaka M. , Miyazaki A.
Published: July 2002

We evaluated the potential prognostic factors for fetomaternal hemorrhage (FMH). Sinusoidal heart rate pattern and decrease in fetal movement are considered important signs of FMH. However these findings were detected in only two of six cases of FMH and were thought to be late signs of FMH. Fetal hemoglobin levels in the maternal circulation for infants born alive following FMH were less than 5%. Our findings suggest that this amount of blood loss could cause fetal death.
Morphology of NADPH‐diaphorase‐positive cells in the retinoceptive layers of the developing rat superior colliculus
Authors: Teno´rio F. , Giraldi‐Guimara˜es A. , Mendez‐Otero R.
Published: July 2002

We have used diaphorase histochemistry to study the morphology of cells expressing nitric oxide synthase in the superficial layers of the superior colliculus of developing and adult rats. The nitric oxide synthase‐positive cells showed a Golgi‐like morphology and were classified according to the cell types identified by several authors using the Golgi method. The first nitric oxide synthase‐positive cells appeared at postnatal day 7 and the number of stained cells increased progressively reaching a maximum at postnatal day 15. The poor staining of the dendritic tree and cell bodies in animals younger than postnatal day 15 allowed no unambiguous identification of the different cell types before that age. At postnatal day 15, based on cell soma and dendritic morphology, we have found that the following cell types express nitric oxide synthase: marginal, horizontal, narrow and wide‐field vertical and stellate. In the adult, the same cell types were found to express nitric oxide synthase but the staining intensity and frequency of each cell type was different from the developing animal. Our results show that cells expressing nitric oxide synthase constitute a subpopulation of neurons in which all cell types are represented. Furthermore, our observations of nitric oxide synthase expression by collicular cells starting by the end of the first postnatal week and reaching a maximum by postnatal day 15 parallels the functional development of the retino‐colliculur and cortico‐tectal projections and suggest that nitric oxide synthase‐positive cells might be involved in this process.
EPR study of the dinuclear active copper site of tyrosinase from Streptomyces antibioticus
Authors: van Gastel Maurice , Bubacco Luigi , Groenen Edgar J.J. , Vijgenboom Erik , Canters Gerard W.
Published: July 2002

The [Cu(I)–Cu(II)] half‐met form of the dinuclear copper site of tyrosinase has been probed by continuous wave electron paramagnetic resonance (EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy in the presence and absence of inhibitors. In all cases the EPR spectrum is indicative of a dx
2−y
2 ground state for the unpaired electron. From the cross‐peaks observed in the HYSCORE spectra, proton hyperfine coupling constants were obtained that are compatible with a hydroxide ion in an equatorial coordination position of the paramagnetic copper. After changing the water solvent to D2O or after addition of the inhibitors p‐nitrophenol or L‐mimosine, the proton signals disappear. The relevance of these findings for understanding the catalytic cycle is discussed.
A potential role for p15Ink4b and p57Kip2 in liver development
Authors: Awad Michael M , Sanders Jennifer A , Gruppuso Philip A
Published: July 2002

Hepatocytes undergo marked changes in proliferation during normal liver development. In order to elucidate the mechanism for these changes, we examined the ontogeny of expression for the known cyclin‐dependent kinase inhibitors (CKIs), p15Ink4b, p16Ink4a, p18Ink4c, p19Ink4d, p21Cip1, p27Kip1 and p57Kip2. All except p16Ink4a were expressed at some time between late gestation and adulthood. The mRNA and protein expression patterns for p15Ink4b and p57Kip2 were consistent with a role for these CKIs in the regulation of hepatocyte proliferation. Specifically, p57Kip2 may contribute to hepatocyte growth arrest that occurs in term fetuses, while p15Ink4b may contribute to the maintenance of adult hepatocytes in a quiescent state. These results assign a possible role to two CKIs not previously identified as involved in hepatocyte cell cycle control.
A novel osteopontin‐like protein is expressed in the trout ovary during ovulation
Authors: Bobe Julien , Goetz Frederick William
Published: July 2002

Using suppression subtraction hybridization between ovulatory and postovulatory trout ovaries, a down‐regulated cDNA was obtained that presumably encodes a novel ovarian protein (‘NOP’). NOP mRNA is present in the ovary during ovulation and down‐regulated by 48 h postovulation, suggesting an important role for NOP during ovulation. Besides the ovary, NOP is also strongly expressed in the testis and at lower levels in the skin, gills, kidney and gastrointestinal tract. While the overall identity is not high, NOP shares several sequence similarities with mammalian and chicken osteopontins, including the percentage of aspartate, serine and alanine residues and the presence of a cell attachment motif.
From p63 to p53 across p73
Authors: Strano Sabrina , Rossi Mario , Fontemaggi Giulia , Munarriz Eliana , Soddu Silvia , Sacchi Ada , Blandino Giovanni
Published: July 2002

In this review we will mainly focus on similarities and differences as well as relationships among p63, p73 and p53.
Effects of FTDP‐17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3β identified by mass spectrometry demonstrate certain mutations exert long‐range conformational changes
Authors: Connell James W , Gibb Graham M , Betts Joanna C , Blackstock Walter P , Gallo Jean-Marc , Lovestone Simon , Hutton Michael , Anderton Brian H
Published: July 2002

In vitro phosphorylation of recombinant wild‐type 2N4R tau and FTDP‐17 exonic mutant forms P301L, V337M and R406W by glycogen synthase kinase 3β (GSK3β) was examined by two dimensional phosphopeptide mapping analysis on thin layer cellulose plates. Comparison of these peptide maps with those generated from wild‐type 1N4R tau isoform from which the phosphopeptide constituents and sites of phosphorylation had been determined previously, enabled us to monitor directly changes in phosphorylation of the individual tau proteins. No differences were found in the phosphorylation of wild‐type, P301L or V337M tau by GSK3β but the R406W mutant showed at least two clear differences from the other three tau proteins. The peptides, identified by mass spectrometry corresponding to phosphorylation at both threonine 231 and serine 235 (spot 3), serines 396, 400 and 404 (spot 6a) and serines 195 and 199 (spot 6b) were absent from the R406W peptide map. The findings imply that the R406W mutation in tau exerts long‐range conformational effects on the structure of tau.
Structural enzymology of biotin biosynthesis
Authors: Schneider Gunter , Lindqvist Ylva
Published: July 2002

Over the last years, significant progress has been made in the understanding of the genetics and enzymology of the biosynthetic pathway of the vitamin biotin. The enzymes catalyzing the last four steps of this pathway, from pimeloyl‐CoA to biotin, provide an ensemble of intriguing reaction mechanisms, which are presently being unravelled. The three‐dimensional structures for three of these enzymes are known and provide a framework to which on‐going mechanistic studies can be related.
Slotoxin, αKTx1.11, a new scorpion peptide blocker of MaxiK channels that differentiates between α and α+β (β1 or β4) complexes
Authors: Garcia-Valdes Jesus , Zamudio Fernando Z , Toro Ligia , Possan Lourival D
Published: July 2002

A novel peptide from Centruroides noxius Hoffmann scorpion venom was isolated and sequenced. The 37 amino acid peptide belongs to the charybdotoxin sub‐family (αKTx1) and was numbered member 11. αKTx1.11 has 75% sequence identity with iberiotoxin and 54% with charybdotoxin. αKTx1.11 revealed specificity for mammalian MaxiK channels (hSlo), thus, was named slotoxin. Slotoxin blocks the MaxiK pore‐forming α subunit reversibly (K
d=1.5 nM). Slotoxin association with α+β (β1 or β4) channels was ∼10 times slower than iberiotoxin and charybdotoxin, leading to a lack of effect on α+β4 when tested at 100 nM for 5 min. Thus, slotoxin is a better tool to distinguish MaxiK α+β complexes.
Micropreparation of peptides by capillary electrophoresis for matrix assisted laser desorption mass spectrometry
Authors: Bergman Ann-Charlotte , Bergman Tomas
Published: July 2002

In the separation of peptides by capillary electrophoresis and analysis by matrix assisted laser desorption mass spectrometry, strong suppression of the mass spectrometric signals is a problem with many common electrolytes used in the separation step such as sodium phosphate. We describe an approach employing individual electrolytes selected for highest performance in each process. Suppression with samples collected into phosphate buffers is avoided when citrate, trifluoroacetic acid or hydrochloric acid is used for collection, while phosphate still provides excellent resolution in the capillary. Low concentrations of hydrochloric acid added to the sample/matrix mixture generate essentially adduct‐free mass spectra with better signal‐to‐noise ratios and detection limits (fmol range) than those obtained with citrate or trifluoroacetic acid. Addition of 0.25% ethylene glycol to both the phosphate electrolyte and the sample improves peak shape and resolution, and is crucial for preparative separations in large diameter capillaries.
Localisation of the C1q binding site within C 1 q receptor/calreticulin
Authors: Stuart G.R. , Lynch N.J. , Lu J. , Geick A. , Moffatt B.E. , Sim R.B. , Schwaeble W.J.
Published: July 2002

Clq receptor (C1gR/collectin receptor) is located on many cell types. Binding of C1q to these cells elicits numerous responses. Protein sequencing has shown that C1gR is almost identical to calreticulin (CaR), an abundant multifunctional protein. Radioiodinated C1gR and CaR bind to C1q with identical characteristics. Three recombinant C1gR/CaR domains (N‐, C‐terminal domains and central P‐domain) were expressed using the Thiofusion system, and used to study the interaction with C1q. Both the N‐ and P‐domains were implicated in C1q binding. A region, termed the S‐domain, spanning the N and P intersection was expressed, and showed concentration‐dependent binding to C1q, demonstrating that the Clq binding site lies within this region.
Effect of polar side chains at position 172 on thermal stability of 3‐isopropylmalate dehydrogenase from Thermus thermophilus
Authors: Akanuma Satoshi , Qu Chunxu , Yamagishi Akihiko , Tanaka Nobuo , Oshima Tairo
Published: July 2002

To understand the role of the amino acid residue at position 172 in the conformational stability, four mutant enzymes of Thermus thermophilus 3‐isopropylmalate dehydrogenase in which Ala172 was replaced with Asp, Glu, Asn, and Gln were prepared by site‐directed mutagenesis. Three mutants were more stable than the wild‐type enzyme. No significant change in catalytic properties was found in the mutant enzymes. The molecular modeling studies suggested that the enhanced thermostability of the mutant enzymes resulted from the formation of extra electrostatic interactions and/or improvement of hydrophobic packing of the interior core.
Resistance to apoptosis in Fanconi's anaemia
Authors: Monti Daniela , Macchioni Sabrina , Guido Marcello , Pagano Giovanni , Zatterale Adriana , Calzone Rita , Cossarizza Andrea , Straface Elisabetta , Malorni Walter , Franceschi Claudio
Published: July 2002

Fanconi's anaemia (FA) is a rare autosomal recessive disease characterised by progressive pancytopoenia, a diverse assortment of congenital malformations, an increased sensitivity to reactive oxygen species and a predisposition to the development of malignancies. In the present study, we assessed the propensity to undergo apoptosis of peripheral blood mononuclear cells (PBMC) from Italian FA patients. Cells were challenged by 2‐deoxy‐d‐ribose (dRib) or TNF‐α plus cycloheximide as agents that induce apoptosis by interfering with cell redox status and mitochondrial membrane potential (MMP), and PBMC from FA patients resulted to be less prone to die than those from healthy subjects. The decreased susceptibility of FA cells to undergo apoptosis was also evident when another parameter highly correlated with the apoptotic process, i.e. MMP, was measured. Moreover, when N‐acetylcysteine was added to dRib‐treated PBMC, a strong protection was evident either in PBMC from control subjects or from FA patients. These data indicate that an alteration of unknown nature of the mechanisms favouring apoptosis is present in freshly collected cells from FA patients, and that such alteration could contribute to the pathogenesis of the disease, and particularly to the increased susceptibility to cancer.
Differential expression of isoforms of PSD‐95 binding protein (GKAP/SAPAP1) during rat brain development1
Authors: Kawashima Nozomu , Takamiya Kogo , Sun Jie , Kitabatake Akira , Sobue Kenji
Published: July 2002

PSD‐95/SAP90, which binds to the C‐terminus of NMDA receptor and Shaker‐type potassium channel, is one of the major postsynaptic density proteins. Recently, novel classes of proteins interacting with the guanylate kinase domain of PSD‐95 have been identified, guanylate kinase‐associated protein (GKAP) and SAP90/PSD‐95‐associated proteins (SAPAPs). Here we report the isolation of new isoforms of PSD‐95 binding protein (GKAP/SAPAP1) using the yeast two‐hybrid system. The isolated protein directly interacts with the guanylate kinase domain of PSD‐95. Northern blot analyses revealed that the expression of these isoforms containing distinct N‐terminal sequences is differentially regulated during brain development. The present findings suggest that each isoform of the PSD‐95 binding protein is differentially expressed in a development‐dependent manner and may be involved in the complex formation of PSD‐95 and channel/receptors at the postsynaptic density.
Yeast gene YOR137c is involved in the activation of the yeast plasma membrane H+‐ATPase by glucose
Authors: de la Fuente Natalia , Maldonado Ana M , Portillo Francisco
Published: July 2002

Glucose triggers transcriptional and post‐transcriptional mechanisms that increase the level and activity of Saccharomyces cerevisiae plasma membrane H+‐ATPase. We have studied the post‐transcriptional activation of the enzyme by glucose and have found that the YOR137c gene product is implicated in this activation. Deletion of YOR137c does not affect the level of Pma1 at the plasma membrane, but disturbs the glucose‐triggered V
max increase of the enzyme. We propose that at least two independent mechanisms are involved in glucose activation of the H+‐ATPase.
Inhibition of chemotactic motility and trans‐endothelial migration of human neutrophils by sphingosine 1‐phosphate
Authors: Kawa Shigeyuki , Kimura Satoshi , Hakomori Sen-itiro , Igarashi Yasuyuki
Published: July 2002

In previous studies, we reported that sphingosine 1‐phosphate (Sph‐1‐P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph‐1‐P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL‐8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans‐endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph‐1‐P specifically inhibited the IL‐8‐ or fLMP‐induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph‐1‐P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph‐1‐P inhibited trans‐endothelial migration and invasiveness of neutrophils into HUVEC‐covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph‐1‐P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph‐1‐P, or its analogs, as anti‐inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.
NO catastrophes in vivo as a result of micellar catalysis
Authors: Gordin Vladimir A. , Nedospasov Andrei A.
Published: July 2002

Micellar catalysis plays a crucial role in NO metabolism because media in vivo are heterogeneous and the concentration of NO in different phases at different levels of solubility differs by degrees of magnitude. The relative volumes of the hydrophobic phases are usually small. At small volumes (which are calculated) of these phases the reaction rates of NO metabolism change. The dependence on the relative volumes is resonance‐like. Not only regulation, but bifurcations and catastrophes are possible in vivo as a result of this changing due to the small change of effectiveness of micellar catalysis.
Expression of skeletal muscle‐type voltage‐gated Na+ channel in rat and human prostate cancer cell lines
Authors: Diss J.K.J. , Stewart D. , Fraser S.P. , Black J.A. , Dib-Hajj S. , Waxman S.G. , Archer S.N. , Djamgoz M.B.A.
Published: July 2002

Previous electrophysiological work has demonstrated expression of a voltage‐gated Na+ channel (VGSC) specifically in two highly metastatic prostatic epithelial tumour cell lines: MAT‐LyLu (rat) and PC‐3 (human). However, the identity of the channel(s) present was uncertain. The present study used a combination of molecular biological techniques to demonstrate that full‐length skeletal muscle type 1 (SkM1) VGSC mRNA is present in the mRNA pool of the MAT‐LyLu cell line. mRNA for this particular channel type was also expressed in the PC‐3 cells. In situ hybridisation data suggested that the level and pattern of rSkM1 mRNA expression were different in the Dunning cells of markedly different metastatic potential. Interestingly, the same type of mRNA was also detected in the weakly metastatic counterparts of the cells: AT‐2 (rat) and LNCaP (human).
β Structure motif recognition by anti‐gliadin antibodies in coeliac disease
Authors: Alfonso Patricia , Soto Claudio , Albar Juan Pablo , Camafeita Emilio , Escobar Héctor , Suárez Lucrecia , Rico Manuel , Bruix Marta , Méndez Enrique
Published: July 2002

A 20‐amino acid synthetic peptide from the N‐terminal region of γ3 avenin yields a surprisingly strong reactivity with anti‐gliadin antibodies (AGA) of coeliac sera, comparable to that of a gliadin extract. In contrast, a low reactivity is observed with five similar peptides derived from α‐gliadin, γ70 and ω1 secalins. Circular dichroism studies of these peptides show that the avenin peptide displays the highest β‐turn content (30%), while other peptides yield much lower values. In agreement with circular dichroism data, nuclear magnetic resonance data point to the presence of a β‐turn in the avenin peptide DPSEQ segment, a sequence with a high statistical β‐turn preference. A strong linear dependence between AGA reactivity and β‐turn content was observed for these peptides, indicating for the first time a role of β‐turn motifs in anti‐gliadin antibodies recognition in coeliac disease. This suggests that circulating AGA in coeliac patients comprise not only linear but also conformational antibodies against β‐turn motifs. Polyclonal antibodies raised against the avenin peptide containing β‐turn motifs react by immunoblotting with all gliadin, hordein and secalin proteins, which are rich in β‐turn conformations, despite that their primary structures are unrelated to that of the peptide.
BEN1 and ZEN1 cDNAs encoding S1‐type DNases that are associated with programmed cell death in plants1
Authors: Aoyagi Shigemi , Sugiyama Munetaka , Fukuda Hiroo
Published: July 2002

We describe the isolation and characterization of cDNAs encoding two DNases that may be involved in the programmed death of plant cells: a 35‐kDa nuclease of barley, which had been reported to be secreted from the aleurone layer into the endosperm during germination and may be responsible for the digestion of nuclear DNA in the course of degradation (cell death) of endosperm, and a 43‐kDa nuclease of zinnia, which appears transiently in association with differentiation to tracheary elements and is likely to participate in the autolysis at the final step of the differentiation. Genes for these nucleases of barley and zinnia were designated BEN1 and ZEN1, respectively. The amino acid sequence of BEN1 protein deduced from the nucleotide sequence of BEN1 cDNA consisted of 288 residues with a putative signal sequence of 23 residues. RNA gel blot analysis revealed that BEN1 mRNA increased in the embryo‐less half seeds of barley in response to the application of gibberellic acid. The deduced amino acid sequence of ZEN1 protein consisted of 303 residues with a putative signal sequence of 25 residues. Temporal accumulation of ZEN1 mRNA was detected during transdifferentiation of zinnia mesophyll cells into tracheary elements. Significant similarities were found among the amino acid sequences of BEN1, ZEN1, nuclease S1 from Aspergillus oryzae, and two other S1‐type nucleases.
A receptor for the lipocalin placental protein 14 on human monocytes
Authors: Miller Robin E , Fayen John D , Chakraborty Shikha , Weber Matthew C , Tykocinski Mark L
Published: July 2002

Human placental protein 14 (PP14), a member of the lipocalin structural superfamily, is an abundant amniotic fluid glycoprotein with documented immunoinhibitory activities. While receptors have been characterized for several other lipocalins, none have been reported to date for PP14. In the present study, two‐color immunofluorescence and flow cytometry was used to screen peripheral blood mononuclear cell subpopulations for their capacity to engage fluoresceinated recombinant PP14. The tagged PP14 bound strongly in a specific and saturable fashion to CD14+ (monocyte lineage) cells, but not to CD20+ (B cell lineage) or CD3+ (T cell lineage) cells. This binding was both pH‐ and temperature‐sensitive, and was reduced by proteolytic pre‐digestion of the cells with trypsin or proteinase K. Scatchard analysis demonstrated a single class of receptors on CD14+ cells, with a K
D of ∼1×10−8 and ∼10–35 000 receptors per cell. These findings constitute the first report of a cell surface‐associated binding protein for PP14 and set the stage for exploring the molecular mechanisms of PP14‐mediated signaling and immunomodulation.
Regulation of the fibronectin EDA exon alternative splicing. Cooperative role of the exonic enhancer element and the 5′ splicing site
Authors: Muro A.F. , Iaconcig A. , Baralle F.E.
Published: July 2002

Alternatively spliced exons generally contain weak splicing sites, and exonic and/or intronic regulatory elements recognised by trans‐acting auxiliary splicing factors. The EDA exon of the fibronectin gene is a typical example of an exon bearing a purine‐rich exon splicing enhancer (ESE) element recognised by members of the SR phosphoprotein family. The regulatory region that governs splicing in the human EDA exon also contains an exon splicing silencer (ESS) element. We have cloned the mouse EDA genomic region, and we show that the ESE and the ESS elements, although they have base differences, can be replaced by the human elements without significant change in the exon inclusion/exclusion ratio. This fact suggests a common splicing regulatory mechanism across species. We demonstrate in vivo the functional activity of the mouse ESE element in splicing. We also show that the trans‐acting factors recognising this element cooperate with the 5′ splicing site of the EDA exon to facilitate proper exon recognition. Indeed, a strong 5′ splicing site overrides the ESE function in exon recognition. However, the presence of a strong 3′ splicing site is not sufficient to compensate for the absence of the splicing enhancer. Our data provide in vivo evidence of the interplay between the exonic splicing regulatory elements and the splicing sites, leading finally to subtle regulation of alternative splicing.
Synthetic lipid products of PI3‐kinase which are added to culture medium prevent low K+‐induced apoptosis of cerebellar granule neurons via Akt kinase activation
Authors: Shimoke Koji , Yamada Masashi , Ikeuchi Toshihiko , Hatanaka Hiroshi
Published: July 2002

To examine which lipid product of phosphatidylinositol 3‐kinase (PI3‐K) is essential for the survival‐promoting pathway in cultured cerebellar granule neurons, three synthetic derivatives of lipid products of PI3‐K were added to culture medium containing a low concentration (5 mM) of potassium (LK+) which induces apoptotic cell death. We found that dipalmitoylphosphatidylinositol 3,4‐bisphosphate and dipalmitoylphosphatidylinositol 3,4,5‐trisphosphate, but not dipalmitoylphosphatidylinositol 3‐monophosphate, effectively blocked the LK+‐induced apoptosis. These two synthetic phospholipids increased Akt activity but not that of PI3‐K. These findings demonstrated that specific lipid products of PI3‐K which are added to culture medium activate Akt/PKB without modulating PI3‐K itself, and as a result prevent neuronal cell death in cerebellar granule neurons.
In the rat, tumor necrosis factor α administration results in an increase in both UCP2 and UCP3 mRNAs in skeletal muscle: a possible mechanism for cytokine‐induced thermogenesis?
Authors: Busquets Sı́lvia , Sanchı́s Daniel , Alvarez Belén , Ricquier Daniel , López-Soriano Francisco J , Argilés Josep M
Published: July 2002

Since the discovery of the new members of the UCP (uncoupling protein) family, UCP2 and UCP3, very few studies have dealt with the regulation of their expression. Bearing this in mind, administration of a single intravenous injection of TNF‐α (100 μg/kg body weight) to rats resulted in a significant increase in UCP2 (242%) and UCP3 (113%) gene expression in skeletal muscle. The results suggest a possible role for UCP2 and UCP3 in the increase of energy expenditure associated with cytokine treatment.
Antiferritin single‐chain antibody: a functional protein with incomplete folding?
Authors: Martsev Sergey P. , Kravchuk Zinaida I. , Chumanevich Alexander A. , Vlasov Alexander P. , Dubnovitsky Anatoly P. , Bespalov Ivan A. , Arosio Paolo , Deyev Sergey M.
Published: July 2002

The pET(scF11) plasmid was constructed comprising the gene of a single‐chain antibody against human ferritin. This plasmid encodes the leader peptide pelB followed by the heavy chain variable VH domain, (Gly4Ser)3 linker peptide, and light chain variable VL domain. The correctly processed scF11 antibody was expressed in Escherichia coli as an insoluble protein without the leader peptide. Purified soluble scF11 was obtained after solubilization in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion‐exchange chromatography. ScF11 demonstrated only a ∼8‐fold decrease in the affinity (K
a=5.1×108 M−1 in RIA and 1.8×108 M−1 in ELISA) vs. the parent IgG2a/κ monoclonal antibody F11. The emission maximum of intrinsic fluorescence strongly suggests a compact conformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein. However, scF11 demonstrated (i) the lack of denaturant‐induced fluorescence ‘dequenching' effect characteristic of the completely folded parent antibody, and (ii) prominent binding, under physiological conditions, of a hydrophobic probe 8‐anilino‐1‐naphthalenesulfonate (ANS) recognizing partially structured states of a protein. These findings are indicative of an incomplete tertiary fold that gives ANS access to the protein hydrophobic core. This work provides the first indication that the functional single‐chain antibody scF11 displays some properties of a partially structured state and therefore may possess incomplete folding.
Generalised bilayer perturbation from peptide helix dimerisation at membrane surfaces: vesicle lysis induced by disulphide‐dimerised melittin analogues
Authors: Takei Jiro , Remenyi Attila , Dempsey Christopher E.
Published: July 2002

The effects of covalent dimerisation of melittin by disulphide formation in cysteine‐substitution analogues, (melittin K23C)2 and (melittin K23Q,Q25C)2, on the kinetics of pore formation in phosphatidylcholine small unilamellar vesicles was measured under low ionic strength conditions. The initial rate of melittin‐induced pore formation increased with the square of the peptide concentration, whereas both disulphide‐dimerised melittin analogues showed a first‐order dependence of pore formation rates on peptide concentration. These results indicate that peptide dimerisation is rate‐limiting for pore formation under these conditions. A model for a generalised bilayer perturbation resulting from the self‐association of a pair of peptide helices at the membrane surface is proposed which may have implications for a number of biological processes that involve the interaction of helical polypeptides with membranes.
Lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte‐macrophage colony stimulating factor
Authors: Tanaka Naohiko , Watanabe Junichi , Kitamura Takayuki , Yamada Yoshitsugu , Kanegasaki Shiro , Nakata Koh
Published: July 2002

Mice deficient in granulocyte‐macrophage colony stimulating factor (GM‐CSF) develop pulmonary alveolar proteinosis (PAP). We found that bronchoalveolar lavage fluid (BALF) from 11 patients with idiopathic pulmonary alveolar proteinosis (IPAP) suppressed the growth of peripheral blood monocytes and TF‐1 cells, a cell line dependent on either GM‐CSF or interleukin‐3 (IL‐3). The inhibitory effect of PAP‐BALF occurred only when TF‐1 cells were cultured with GM‐CSF but not when cultured with IL‐3, suggesting that PAP‐BALF contains a factor that specifically interferes with GM‐CSF function. 125I‐GM‐CSF binding to TF‐1 cells was prevented in the presence of BALF from IPAP patients. Furthermore, cross‐linking of 125I‐GM‐CSF to IPAP‐BALF produced two major bands on SDS‐PAGE; these bands were not observed in normal BALF. These data suggest that IPAP is caused by expression of binding factor(s) which inhibit GM‐CSF function in the lung.
Evolution of visual pigments in geckos
Authors: Taniguchi Yuki , Hisatomi Osamu , Yoshida Masao , Tokunaga Fumio
Published: July 2002

Most geckos are nocturnal forms and possess rod retinas, but some diurnal genera have pure‐cone retinas. We isolated cDNAs encoding the diurnal gecko opsins, dg1 and dg2, similar to nocturnal gecko P521 and P467, respectively. Despite the large morphological differences between the diurnal and nocturnal gecko photoreceptor types, they express phylogenetically closely related opsins. These results provide molecular evidence for the reverse transmutation, that is, rods of an ancestral nocturnal gecko have backed into cones of diurnal geckos. The amino acid substitution rates of dg1 and dg2 are higher than those of P521 and P467, respectively. Changes of behavior regarding photic environment may have contributed to acceleration of amino acid substitutions in the diurnal gecko opsins.
Identification of cold‐sensitive mutations in the Schizosaccharomyces pombe actin locus
Authors: McCollum Dannel , Balasubramanian Mohan , Gould Kathleen
Published: July 2002

In recent years, the actin cytoskeleton in Schizosaccharomyces pombe has become the subject of intense scrutiny. However, to date, only a single actin mutation has been identified. Described here is the isolation and characterization of four new cold‐sensitive actin mutations. Sequence analysis of the mutant actin genes indicated that each of these mutations caused alterations in single amino acids that are conserved in all actin sequences. These mutants differ in their phenotypes. One of these mutations (act1‐48) was identified as an extragenic suppressor of a mutation in the cdc4 gene, which is required for actin ring formation and cytokinesis. Interestingly, when act1‐48 mutant cells were shifted to the restrictive temperature, actin patches were not detected but the actin ring formation and stability was unaffected. The three other mutations, act1‐16, act1‐32 and act1‐67, primarily affected the actin ring formation or stability while F‐actin patches did not seem to be substantially different in appearance. Given that the ultrastructural architectures of F‐actin patches and the F‐actin ring are presently unclear, these mutations, which affect one structure or the other, should be useful for future studies on the role of actin itself in the function of these F‐actin‐containing structures in S. pombe.
Characterization of a novel monoclonal antibody that senses nitric oxide‐dependent activation of soluble guanylate cyclase
Authors: Tsuyama Shingo , Yamazaki Eri , Tomita Takeshi , Ihara Hideshi , Takenaka Shigeo , Kato Keiko , Kozaki Shunji
Published: July 2002

Two monoclonal antibodies (mAbs) against bovine lung soluble guanylate cyclase (sGC) were prepared and characterized. mAb 3221 recognized both the α‐ and β‐subunits of sGC and had greater binding affinity to the enzyme in the presence of NO. mAb 28131 recognized only the β‐subunit and its affinity did not change with NO. Neither mAb cross‐reacted with particulate GC. Cultured Purkinje cells from rats were treated with S‐nitroso‐N‐acetylpenicillamine, an NO donor, and examined by immunocytochemical methods. The immunoreactivity associated with mAb 3221 increased with the cGMP content in a crude extract of cerebellum and the NO2 generated in the culture medium increased.
R‐esp1, a rat homologue of Drosophila Groucho, is differentially expressed after optic nerve crush and mediates NGF‐induced survival of PC12 cells
Authors: Arndt Marco , Bank Ute , Frank Karin , Sabel Bernhard A. , Ansorge Siegfried , Lendeckel Uwe
Published: July 2002

The differential display reverse transcription polymerase chain reaction method was used to detect alterations in gene expression in the superior colliculus after optic nerve crush in adult rats. One of the most prominent changes observed was the selective induction of R‐esp1, a homologue of the Drosophila enhancer of split locus (Groucho). Therefore, we studied the influence of R‐esp1 on nerve growth factor (NGF)‐induced cell survival of PC12 cells. Overexpression of R‐esp1 promotes cell survival even in the absence of NGF and, conversely, it is reduced by antisense‐mediated inhibition of R‐esp1 expression. In conclusion, we propose a novel model in which R‐esp1 protein mediates the NGF‐signaling pathway.
ATPase activity of the heat shock protein Hsp72 is dispensable for its effects on dephosphorylation of stress kinase JNK and on heat‐induced apoptosis
Authors: Volloch Vladimir , Gabai Vladimir L. , Rits Sophia , Sherman Michael Y.
Published: July 2002

A major inducible heat shock protein, Hsp72, has previously been found to stimulate dephosphorylation (inactivation) of stress kinase JNK in heat‐shocked cells and protect them from apoptosis. Using Rat‐1 fibroblasts with constitutive expression of a human Hsp72 or its deletion mutant lacking an ATPase domain (C‐terminal fragment (CTF)), we tested whether ATPase activity of Hsp72 is necessary for these effects. We found that expression of CTF markedly increased, similarly to the intact protein, JNK dephosphorylation in heat‐shocked cells. As a result, JNK inactivation following heat shock occurred much faster in cells expressing either full‐length or mutant Hsp72 than in parental cells and this was accompanied by suppression of heat‐induced apoptosis. Thus, protein refolding activity of Hsp72 appears to be dispensable for its effect on JNK inactivation and apoptosis.
Spectral tuning of a circadian photopigment in a subterranean ‘blind’ mammal (Spalax ehrenbergi)
Authors: David-Gray Zoë K. , Cooper Howard M. , Janssen Jannie W.H. , Nevo Eviatar , Foster Russell G.
Published: July 2002

The atrophied subcutaneous eyes of Spalax ehrenbergi (the blind mole rat) express a long wavelength sensitive (LWS) cone opsin. Our data provide strong evidence that this photopigment is spectrally tuned to enhance photon capture in the red light environment of the eye. Furthermore, novel mechanisms appear partially responsible for this sensory fine‐tuning. These data support the hypothesis that the LWS opsin of Spalax acts as a functional photopigment and that it is not a ‘residue’ of the pre‐subterranean visual system. As the eye of Spalax has only one known function, the entrainment of circadian rhythms to environmental light, the LWS photopigment is implicated in this task. These results, together with our recent findings that rod and cone photopigments are not required for murine photoentrainment, suggest that multiple photopigments (classical and novel) mediate the effects of light on the mammalian circadian system.